Slingshot-1 (SSH1) phosphatase Controls Cytoskeletal Remodeling, Integrin conformation and Metabolic Reprogramming During CD4 T Cell Activation

Slingshot-1 (SSH1) phosphatase Controls Cytoskeletal Remodeling, Integrin conformation and Metabolic Reprogramming During CD4 T Cell Activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Slingshot-1 (SSH1) phosphatase Controls Cytoskeletal Remodeling, Integrin conformation and Metabolic Reprogramming During CD4 T Cell Activation Noa Martí, Alvaro Gómez-Morón, Marta Lozano-Prieto, Camila Scagnetti, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8604514/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Effective T cell activation requires formation of the immunological synapse (IS), which depends on coordinated remodeling of actin and microtubule cytoskeletons. Upon T cell receptor (TCR) engagement, the phosphatase Slingshot-1 (SSH1) is rapidly recruited to the nascent IS, where it dephosphorylates cofilin and suppresses LIM kinase activity to promote actin dynamics. However, the mechanism by which SSH1 anchors at and controls IS formation is unknown. Here, we identify the role of SSH1 in assembling a protein hub with Talin-1, Kindlin-3, ADAP, and Myosin IIA that promotes high-affinity activation of the integrin LFA-1. This allows SSH1 to coordinately regulate actin and microtubule dynamics and ultimately facilitates the metabolic reprogramming of T cells. Loss of SSH1 disorganizes IS in terms of mitochondria, rewiring T cells toward fatty acid oxidation, congregating lipid droplets and peroxisomes, and preventing glucose metabolism. Together, our findings establish SSH1 as a node connecting cytoskeletal dynamics to metabolic adaptation for T cell activation. Biological sciences/Cell biology/Cell adhesion/Integrins Biological sciences/Immunology/Imaging the immune system Biological sciences/Cell biology/Cytoskeleton/Actin Biological sciences/Immunology/Adaptive immunity/Cellular immunity/Lymphocyte activation Biological sciences/Biochemistry/Lipids/Fatty acids Slingshot-1 T lymphocytes metabolism mitochondria cytoskeleton integrin activation Immunological Synapse Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Main T cell activation is a tightly coordinated process that couples cell signaling with dynamic cytoskeletal remodeling. Upon T cell receptor (TCR) engagement by antigen, substantial rearrangements of both the actin and tubulin networks occur at the nascent immunological synapse (IS) (1). These cytoskeletal dynamics are essential for sustaining TCR signaling and driving full T cell effector function (2). A critical outcome of these early cytoskeletal events is the complete activation of the integrin LFA-1 to support cell adhesion to the antigen-presenting cell (APC), a process that requires active actin remodeling at the synapse as well as the interaction of Talin-1, Kindlin-3 and ADAP cytoskeletal adaptors (3,4). Beyond adhesion, LFA-1 engagement triggers broader cellular reprogramming at the IS. LFA-1 ligation provides mechanical and co-stimulatory signals that drive the polarization of intracellular organelles toward the IS. For instance, LFA-1–Erk1/2 signaling promotes centrosome translocation to the IS (5), and LFA-1 activation is required for repositioning of mitochondria to the T cell–APC interface (6). At the IS, mitochondria serve as localized powerhouses and calcium buffers (7), sustaining the high ATP demands and prolonged Ca 2+ signaling needed for full T cell activation (8). In this manner, the convergence of cytoskeletal regulation and organelle positioning has profound consequences for T cell metabolism and cancer immunotherapy (9). Slingshot-1 (SSH1) phosphatase has emerged as a molecular regulator linking TCR signals to actin cytoskeleton remodeling (10). SSH1 is a dual-specificity phosphatase that dephosphorylates and activates cofilin, thereby promoting actin filament turnover (11, 12), and can concomitantly inactivate LIM-kinase (LIMK) to boost cofilin activity (13). SSH1 depletion in CD4 T cells affects CD3ε conformational change and Nck recruitment, which leads to increased actin dynamics and unstable IS, showing disrupted TCR organization and early signaling in a process regulated by LIMK. (10). Nevertheless, it remains unclear whether these actions on actin organization exert other effects on T cell biology through IS regulation. In this regard, TCR and co-stimulatory signals trigger a well-characterized metabolic rewiring in T cells. Quiescent T cells shift to a highly glycolytic state while also boosting mitochondrial oxidative capacity, thereby meeting the increased energetic and biosynthetic demands of clonal expansion, differentiation and effector function (14, 15). Here, we show that SSH1 orchestrates the remodeling of both actin and microtubule networks to ensure optimal CD4 + T cell activation and metabolic reprogramming. Mechanistically, SSH1 is required for the molecular association of ADAP and Myosin-IIA in a multiprotein complex containing Talin-1 and Kindlin-3 that enables LFA-1 to achieve its fully active conformation and properly localize at the IS. This allows SSH1 to regulate the repositioning of mitochondria, peroxisomes, and lipid droplets at the IS through coordinated actin–microtubule crosstalk. Consequently, SSH1-deficient CD4 + T cells show dysregulated mitochondria and peroxisomes activities, including marked ROS production and lipid peroxidation upon IS formation. Collectively, our findings establish SSH1 role in linking adhesion and cytoskeletal dynamics to organelle positioning to facilitate metabolic remodeling and to drive efficient CD4 + T cell antigenic responses. Results SSH1 nucleates Talin-1, Kindlin-3, ADAP and Myosin IIA at the IS SSH1 is activated by TCR triggering and regulates actin cytoskeleton remodeling by its phosphatase activity on cofilin and LIMK, with SSH1-deficient cells showing increased actin dynamics and capacity to extend motile lamellae (10). To address the way SSH1 localizes at the IS and how it may propagate the TCR signaling, we first searched for potential interactors between SSH1 and key actin regulators at the IS through confocal microscopy. JK T cells were conjugated with SEE-loaded Raji B cells, acting as APCs to establish the IS. SSH1 co-distributed at these ISs with F-actin and Talin-1 (Fig. 1a) . The profiles for the distribution of the mean fluorescence intensity (MFI) of these components in 3D-IS modeling revealed that SSH1 accumulated mainly at the external area of the IS, with a similar pattern to Talin-1 and F-actin (Fig. 1a) . Talin-1 connects actin dynamics to LFA-1 integrin (16), which may serve to help SSH1 localization at the IS and the motile lamellae observed in T cells expressing low or no SSH1 (17). Next, human primary CD4 T cells forming synapse-like structures with beads coated with stimulating anti-CD3 and anti-CD28 antibodies were studied. SSH1 co-localized with Talin-1 ( Fig. 1b,e ), Kindlin-3 ( Fig. 1c,e ) and ADAP/Fyb ( Fig. 1d,e ) at the T cell side of the contact. The molecular association between SSH1, Talin-1, Kindlin-3 and ADAP was confirmed through co-immunoprecipitation (co-IP) assays in resting and stimulated JK T cells (Fig. 1f,g) . TCR triggering enhanced the interaction of SSH1 with these proteins. Talin-1 and kindlin-3 mainly interacted with each other upon TCR activation and with the other members of the complex. ADAP was the member of the complex that showed more restrictive interaction in resting conditions and showed inducible capacity to co-IP with the other members of the complex upon TCR activation. These proteins are involved in the complete activation of LFA-1 integrin by propagating TCR signals to the integrin activation machinery (18-20). Indeed, Talin-1 and Kindlin-3 complex regulates the action of Myosin IIA (21), which is required to organize mechanical forces at the IS (22). Therefore, the presence of Myosin-IIA was probed in SSH1 complexes, finding that their association was highly increased upon TCR stimulation ( Fig. 1h,i ). Indeed, protein-protein associations were observed for Myosin IIA, Talin-1, Kindlin-3 and ADAP upon TCR triggering and co-stimulation ( Fig. 1j ), with ADAP and especially Talin-1 showing major recovery of the complex upon activation ( Fig. 1j,k ). When Myosin IIA was immunoprecipitated, it apparently associated with the other proteins comparably in resting and activated cells ( Fig. 1j,k ). To investigate whether this protein network depended on SSH1, similar experiments were performed in cells lacking SSH1 ( SSH1 KO CRISPR/Cas9 JK T cells). In the absence of SSH1, ADAP was unable to properly incorporate in the protein complex, with low or no recovery of Talin-1, Kindlin-3 and Myosin-IIA ( Fig. 1l,m ). ADAP was completely absent from the protein network when Talin, Kindlin-3 or Myosin IIA were immunoprecipitated.However, the interaction between Talin-1 and Kindlin-3 was conserved, although Myosin IIA was not recovered anymore in the complex ( Fig. 1l,m ). In contrast, Myosin IIA seemed able to recover Kindlin-3 and Talin-1 irrespective of stimulation in the absence of SSH1 ( Fig. 1l,m ). These results suggest a potential complex where SSH1 would help Talin-1/Kindlin-3 complex to connect with ADAP and Myosin-IIA, and that this complex could be induced upon TCR activation and formation of the IS. Therefore, SSH1 seems to bring together active molecules to the F-actin structures to regulate cell adhesion ( Fig. 1n ). In support of this, the phosphorylation of the regulatory myosin light chain (MLC) at pT18 and pS19 was decreased in siSSH1 CD4 T cells ( Fig. 1o,p ). This phosphorylation is a key event for Myosin IIA activation and tension generation (23), which points to SSH1 as potential regulator of Myosin-IIA activity at the IS. SSH1 may therefore serve as a scaffold to recruit connectors of F-actin and membrane receptors, as can be integrins, to help the stability of T-APCs contacts. LFA-1 requires SSH1 to adopt its active conformation To gain insight into the potential role of a SSH1-dependent complex including proteins that regulate the activation of LFA-1 integrin (24,25), we investigated SSH1 requirement for specific active, conformational state of LFA-1 ( Fig. 2a ). Specific extended-closed and extended-open conformational epitopes on LFA-1 were detected through two specific monoclonal antibodies, KIM127 and m24, respectively (26) (Fig. 2a) .Human primary CD4 T cells SSH1 expression was targeted by small interfering RNAs (siRNAs) against the SSH1 gene (siSSH1) or unspecific siRNA (siCTRL) ( Extended Data Fig. 1a). Cells were allowed to spread over anti-CD3 and anti-CD28 antibodies-coated plates and analyzed through flow cytometry. Activated siSSH1 CD4 T cells contained decreased extended-closed (Fig. 2b) and extended-open (Fig. 2c) LFA-1, which was also confirmed in SSH1 KO JK T cells ( Extended Data Fig.1b,c ). In contrast, VLA-4 activation was unaffected in these cells in similar assays by using HUTS21 antibody, which recognizes an activation-dependent epitope of VLA-4 integrin (27) ( Extended Data Fig.1d,e ). The expression of LFA-1 and VLA-4 integrins remained unchanged by loss of SSH1 ( Extended Data Fig.1f ). The effect of SSH1 absence was explored in depth by studying the spatial distribution of total and active LFA-1 at the IS. In control cells, the majority of detected LFA-1 showed extended-closed conformation and was found at the IS (Fig. 2d) , in the internal side of the F-actin ring ( 3D in Fig. 2e) , while low extended-close LFA-1 was observed in SSH1 defective cells, with an aberrant localization of LFA-1 at more external areas of the F-actin ring (Fig. 2d,e) . To confirm the aberrant localization of LFA-1 integrin at the IS regarding the F-actin ring, specific quantifications were performed in control and SSH1 KO JK T cells, finding increased LFA-1 co-localizing with F-actin in SSH1 KO cells ( Extended Data Fig.1g-j) . In addition, in control cells, the more active, extended-open LFA-1 was found at more internal localizations of the F-actin ring, with low LFA-1 molecules stained with the specific antibody, while SSH1 KO cells showed active LFA-1 localized at F-actin regions (Fig. 2f,g). These data point to decreased adhesion in the absence of SSH1. In this regard, although VLA-4 activation was not affected when studied through flow cytometry ( Extended Data Fig.1d,e ), its active form showed an altered distribution in the synapse ( Extended Data Fig.1k,l ), suggesting a general role for SSH1 in integrin localization at the IS upon activation, probably through promoting the transmission of the inside-out signaling from the TCR to the cytoskeleton and facilitating the local, contractile activity of Myosin IIA. In support of this, the phosphorylation and activation of Pyk2 pY402 and Vav1 pY174 were decreased in siSSH1 CD4 T cells (Fig. 2h,i ) and SSH1 KO JK T cells, which showed similar defects in Myosin-IIA activation by means of decreased myosin light-chain phosphorylation, as observed for siSSH1 cells ( Extended Data Fig.1m,n ). Vav1 and Pyk2 are relevant mediators for inside-out signaling by connecting TCR to actin cytoskeleton remodeling and integrin clustering and by regulating the position of the centrosome at the IS, which may be also regulated by actin dynamics (1,28). SSH1 promotes tubulin dynamics after TCR activation Actin and microtubules are both critical for integrin activation, which confer stability to the synapse, and force transmission through ADAP and Myosin-IIA (29,30). This can be required to move the centrosome, together with proper control of F-actin at the centrosome area (31), which could be altered by loss of SSH1. In this regard, primary siSSH1 CD4 T cells did not polarize their centrosome to IS (Fig. 3a,b). We therefore studied whether centrosomal actin in JK-Raji conjugates was correctly regulated in SSH1 KO cells (Fig. 3c) , which showed a decreased centrosome polarization index (Fig. 3d) ,concomitant with an increased centrosomal F-actin index (Fig. 3e) . This resulted in the loss of correlation between both indexes (Fig. 3f) compared with control JK T cells (Fig. 3g) . SSH1-silenced primary CD4 T cells also showed increased F-actin surrounding the centrosome (PCM-1 centered in tubulin in maximal projections) upon activation, which can be observed in 3D together with a defect in the clearance of actin at the central part of the IS (Fig. 3h). siSSH1 CD4 T cells showed increased F-actin index at the centrosomal area (Fig. 3i ). Indeed, this F-actin seems to arise from the non-polarized Golgi complex, which surrounds the centrosome, in siSSH1 CD4 T cells, connecting Golgi with the IS area (Fig. 3h , j). Golgi distance to the IS was consequently increased in IS formed by SSH1 KO JK T cells with APCs ( Extended Data Fig. 2a,b ). In SSH1-defective cells, the excess of F-actin at the centrosomal area may interfere with the localization of the centrosome at the IS, preventing its relocation toward the IS. Since the orientation of the centrosome can rescue defective centrosomal polarization in terms of the organization of the microtubule network at the IS (32),westudied upstream signaling for microtubule regulation such as LIMK1 and Aurora kinase A, which is upstream of PKC during T cell activation and required for centrosomal positioning (33). Cells defective for SSH1, either JK or primary CD4 T cellsshowed increased phosphorylation of LIMK1/2 pT508/505 (Fig. 3k,l, Extended Data Fig. 2c,d ), as well as Aurora A phosphorylation at T288 (Fig. 3k,l, Extended Data Fig. 2c,d ). To assess tubulin dynamics at the IS, live-cell total internal reflection fluorescence microscopy (TIRFm) was used to track EB3-GFP decorated plus-tips of microtubules in SSH1 KO JK T cells during IS. Control cells were able to polarize the centrosome near the IS in these assays while SSH1 KO JK T cells showed reduced microtubule dynamics (Fig. 3m,n, Supplementary Video 1) . This was reflected by the loss of acetylation kinetics of α-tubulin at lysine 40 (K40-α-tubulin) upon IS formation ( Fig. 3o) , that did not decrease rapidly upon TCR activation as described before (34) and increased in cells forming synapses for extended times ( Extended Data Fig. 2e,f ). Therefore, these data point to SSH1 as a regulator of actin dynamics at different locations in activated cells, connecting actin and microtubule dynamics at the IS and helping the intracellular organization of T cells. Metabolic regulation of T cells is fine-tuned by SSH1 After TCR engagement, mitochondria are transported along microtubules and accumulate around the translocated centrosome and Golgi at the IS, beneath the F-actin ring in a process also regulated by LFA-1 integrin adhesion (6,35-37). There, mitochondria fuel myosin light-chain phosphorylation (38). Therefore, mitochondria, LFA-1 and F-actin were studied through confocal microscopy at the IS established by control and SSH1 KO JK T cells with SEE-loaded Raji cells, finding defective mitochondria positioning at the IS in the absence of SSH1 (Fig. 4a,b ). Likewise, siSSH1 CD4 T cells were unable to polarize mitochondria to the IS (Fig. 4c,d, Extended Data Fig. 3a). To further assess mitochondria in these cells, we isolated mitochondria from resting or stimulated control and SSH1 KO JK T cells to study their attached regulatory, cytoskeletal and motor components, such as kinesin-1 and cytosolic dynein (Fig. 4e,f). Mitochondrial-resident SSH1 was found increased in activated control JK T cells together withLIMK, as well as α-tubulin and β-actin, which were increased in these mitochondria, reflecting their relationship with the cytoskeleton to organize their movement to the IS. In addition, LIMK1/2 seems activated, as it is phosphorylated at pT508/505, and tubulin shows acetylation at Lys 40, indicating stability of microtubules ( Fig. 4e) . The lack of SSH1 increased LIMK1 recruitment, as well as acetylation of tubulin and the amount of actin recovered ( Fig. 4e ), suggesting increased stability of the cytoskeleton around the mitochondria, and augmented docking of the mitochondria in these cells. In this regard, in control cells TCR activation induced the binding of kinesin-1 molecular motor, formed by kinesin heavy chain (KHC) and kinesin-1 light chain (KLC), and moderate recruitment of p74-dynein (cytosolic dynein) (Fig. 4f) . Kinesin-1, a molecular motor leading movement toward the plus-end of microtubules (36,39), would facilitate mitochondrial transport along microtubules toward the plasma membrane to reach the IS, whereas dynein would transport mitochondria toward the centrosome. In SSH1 KO JK T cells, the kinesin-1 was greatly increased upon activation after T cell stimulation, as well as dynein (Fig. 4f) . Taken together, these results suggest that SSH1 regulation of actin and tubulin cytoskeleton is required to regulate the interaction of mitochondria with the cytoskeleton and the proper recruitment of the molecular motors involved in their transport. This transport along the cytoskeleton is essential to position mitochondria correctly at the IS (36,39), which is not observed in SSH1-defective cells. Beyond localization, the fate and function of mitochondria depend on cytoskeletal docking (36,39). Therefore, mitochondrial mass was assessed by staining cells with NAO, a mitochondrial probe that binds to cardiolipin in mitochondria, independently of the mitochondrial membrane potential (ΔΨm). When compared with control cells, no differences were observed in siSSH1 CD4 T cells (Fig. 4g) , although SSH1 KO JK T cells exhibited a decrease in mitochondrial mass ( Extended Data Fig. 3b ). Mitochondrial ROS was then analyzed, finding that siSSH1 CD4 T cells produced higher amounts of ROS, which were further increased shortly upon T cell activation (5 min) and sustained for several hours, while siCTRL cells showed a more modest increase at 15 min, that dropped in the first hour of activation ( Fig. 4h, Extended Data Fig. 3c) . These results indicate that the mitochondrial complexes required for oxidative phosphorylation (OXPHOS) are not correctly responding in SSH1-deficient cells. ΔΨm was then analyzed with MitoTracker Deep Red FM normalized to Tom20 in siSSH1 CD4 T cells, which were hyperpolarized, whereas control cells exhibited a drop in ΔΨm at 2-15 min after T cell activation, which corresponds to high production of ATP early after TCR activation (39), and then a re-polarization upon 30 min of stimulation ( Fig. 4i, Extended Data Fig. 3d) . Increase in ΔΨm was corroborated through confocal microscopy in siSSH1 CD4 T cells ( Fig. 4j-l ) and SSH1 KO JK T cells ( Extended Data Fig. 3e,f ) by measuring MitoTracker Orange/Tom20 ratios . To further address mitochondria activity, control and siSSH1 CD4 T cells were subjected to a mitostress test using glucose as external energy source and measuring the oxygen consumption rate (OCR) as indicator of mitochondrial respiration (Extended Data Fig. 3g) . siSSH1 CD4 T cells showed unchanged basal respiration, with decreased ATP production after TCR stimulation compared with siCTRL CD4 T cells ( Fig. 4m) , corroborating our data on mitochondrial hyperpolarization. In addition, inaccordance with the defects observed in the recruitment of the required molecular motors to help reorganize the IS in activated T cells, siSSH1 CD4 T cells did not respond to TCR stimulation by increasing their maximal respiration and spare respiratory capacity as did siCTRL cells ( Fig. 4m ). In the case of SSH1 KO JK T cells, an exacerbated phenotype was observed, with decreased basal respiration, ATP production and maximal respiration at resting conditions and in response to TCR activation ( Extended Data Fig. 3h,i ), probably due to supplementary problems due to long-term knock-down of SSH1, such as decreased mitochondrial mass ( Extended Data Fig. 3b ). siCTRL and siSSH1 CD4 T cells were also challenged to ascertain whether they were able to oxidize glucose through glycolysis, which is known to increase during T cell activation (14). Extracellular acidification rate (ECAR) was used as an indicator of glycolysis by using the Seahorse analyzer (Extended Data Fig. 3j) . siSSH1 CD4 T cells showed reduced capacity to increase glycolysis and glycolytic capacity upon stimulation with anti-CD3 and anti-CD28 antibodies, with no significant changes without TCR stimulation ( Fig. 4n) . SSH1 KO JK T cells exhibited reduced glycolysis and glycolytic capacity both at rest and after TCR stimulation ( Extended Data Fig.3k,l ). A reduction in the Akt/mTOR/S6 signaling pathway was observed in siSSH1 CD4 T cells, although significant signaling levels were still present with phosphorylation of Akt, mTOR and S6 ( Fig. 4o,p ). This decrease was also observed in SSH1 KO JK T cells ( Extended Data Fig. 3m,n ) after T cell activation, pointing to reduced response to TCR activation and co-stimulation. SSH1 connects organelle localization and metabolic response Although Akt/mTOR signaling can be detected in CD4 T cells expressing low levels of SSH1, their metabolic response seems highly affected. An emerging hypothesis indicates that inter-organelle contacts collaborate in organizing T cell metabolic responses (9). To gain insight into this possibility, the distribution of mitochondria and peroxisomes regarding the tubulin cytoskeleton during IS formation was assessed. Whereas siCTRL CD4 T cells showed re-localization of the centrosome, mitochondria and peroxisomes to the IS, siSSH1 CD4 T cells exhibited defective localization of peroxisomes at the IS ( Fig. 5a,b) . SSH1 KO JK CD4 T cells paralleled the redistribution found in siSSH1 CD4 T cells during IS ( Fig. 5c, Extended Data Fig. 4a) . siSSH1 CD4 T cells exhibited a phenotype similar to unstimulated CD4 T cells conjugated to control beads, with their mitochondria and peroxisomes distributed throughout the cell ( Extended Data Fig. 4b ). The number of peroxisomes was increased in siSSH1 CD4 T cells ( Fig. 5d,e ), as well as in SSH1 KO JK T cells ( Extended Data Fig. 4c-e ). In addition, peroxisomes proximity to mitochondria increased in siSSH1 CD4 T cells, showing augmented co-localization ( Fig. 5d,f ), with similar results in SSH1 KO JK T cells (Extended Data Fig. 4c,f ). Further investigation by flow cytometry also showed increased peroxisomes in SSH1 KO JK T cells ( Fig. 5g, Extended Data Fig. 4g ). Since peroxisomes regulate β-oxidation of long-chain fatty acids until they can be oxidized by mitochondria (40), the increased interaction between mitochondria and peroxisomes and the increase in peroxisomes point to a shift to fatty acid catabolism in SSH1 defective cells. To probe this hypothesis, neutral lipids were studied by Bodipy 493/503 probe through flow cytometry in SSH1 KO JK T cells. These cells exhibited higher MFI, suggesting increased neutral lipids in cells ( Fig. 5h ), the main component of lipid droplets (LD), composed of a core of neutral lipids surrounded by a single layer of phospholipids (41). These results, together with the increased peroxisomes ( Fig. 5g ) and mitochondrial ROS production ( Fig. 4h ) in SSH1-deficient CD4 T cells, prompted the study of lipid peroxidation in these cells. We used a specific probe that changes its emission wavelength from ∼590 nm to ∼510 nm when neutral lipids are peroxidized. These assays revealed that lipid peroxidation increased in SSH1 KO JK T cells ( Fig. 5i, Extended Data Fig.4h ). LD localization, number and proximity to mitochondria and peroxisomes, known to establish inter-organelle contacts (42), were studied in CD4 T cells and in JK CD4 T cells through confocal microscopy. SSH1 depletion increased the number of LDs per cell ( Fig. 5j,k, Extended Data Fig.4i,-k), as well asthe number of LD contacts with peroxisomes and mitochondria ( Fig. 5j,l,m, Extended Data Fig.4i,l,m) , consistent with defective polarization of LDs to the IS formed by SSH1 KO JK T cells and SEE-loaded Raji B cells (Extended Data Fig. 4j,n ). This supports a differential use of LDs in SSH1-silenced cells. Therefore, endogenous fatty acid oxidation (FAO) was determined in siCTRL and siSSH1 CD4 T cells. CD4 T cell activation induced FAO, and defective SSH1 expression led to a 50% increase in FAO in resting conditions, which is further reinforced in response to TCR and co-stimulation ( Fig. 5n, Extended Data Fig. 4o-p ). This response was enhanced when palmitate was provided as external source in similar assays, showing a 100% increase in FAO in resting conditions, further augmented by activation in siSSH1 CD4 T cells ( Fig. 5o, Extended Data Fig. 4p ). SSH1 KO JK T cells showed a similar increase in FAO, under basal conditions and following TCR stimulation, both during oxidation of endogenous fatty acids and in the presence of exogenous palmitate ( Extended Data Fig. 4q-t ). In addition, SSH1-silenced cells showed sustained OCR when etomoxir was injected, while OCR fell dramatically in control cells (Extended Data Fig. 4u ). This is consistent with carnitine palmitoyltransferase I (CPT1)-independent FAO pathways, such as peroxisomal β-oxidation, and aligns with our data of lipid peroxidation ( Fig. 5i ). This suggests an augmented peroxisomal FAO capacity due to the observed increased LD-peroxisome-mitochondria contacts and peroxisome number. These data demonstrate that SSH1 controls organelle distribution and localization at the IS, helping mitochondrial respiration and glycolysis shift, and that SSH1 absence enhances lipid metabolism through LD increase and regulation of their contacts with peroxisome and mitochondria ( Fig. 6 ). Discussion This study identifies SSH1 phosphatase as a central coordinator of actin and tubulin cytoskeleton crosstalk and LFA-1 integrin activation through the establishment of a protein hub facilitating proper organelle positioning during T cell activation. Hence, our data show that SSH1 bridges key integrin regulators Talin-1, Kindlin-3 and ADAP (18, 20) at the IS and facilitates the transition of LFA-1 into its high-affinity state ( Fig. 6). Talin-1, Kindlin-3 and ADAP, stabilize high-affinity LFA-1 clusters at the IS (19) allowing transduction of TCR signals to integrin activation, helping inside-out signaling (18). In this context, the described SSH1-driven actin severing and turnover (11) may promote Talin-1 binding and tensile actin flow that separate the LFA-1 integrin α/β tails, which is the final inside-out step of LFA-1 integrin activation (43, 44). The described complex in this study extends the role of SSH1 in the control of T cell actin remodeling, since SSH1 allows Talin-1 and Kindlin-3 to interact with ADAP and Myosin IIA, ultimately promoting complete LFA-1 integrin activation and localization, mainly at the more internal area of the F-actin ring formed in the lamella of T cells organizing IS. There, SSH1-deficient cells showed reduced LFA-1 in extended-closed, and especially in extended-open conformations. These findings underscore SSH1 as a key molecule for building high-avidity LFA-1 adhesions at the synapse. In this regard, actin-driven forces are known to stabilize high-affinity LFA-1, and tensile stress orients the integrin on the membrane and locks its headpiece open (45). Our results show that SSH1 and Myosin IIA form part of a regulated complex that is fostered by T cell activation via TCR, bringing together Talin-1, Kindlin-3 and ADAP with Myosin IIA activity. These data suggest that SSH1 acts as a scaffold protein, maintaining the complex hub for integrin activation and the inside-out integrin TCR-derived signaling required for this activation, as observed in the case of phosphorylation and activation of Pyk2 (pY402) and Vav1 (pY174), and Myosin IIA through the phosphorylation of myosin regulatory light chain (MLC2) pT18/pS19, required for tension generation and mechanotransduction (22-24). This action seems specific to LFA-1, since VLA-4 integrin activation is not affected in SSH1-deficient T cells. This could be explained because of the differential location of these integrins at the IS. VLA-4 integrin acts distally, while LFA-1 acts at the lamella. In this regard, beta-2 integrins, such as LFA-1, in contrast to beta-1 integrins, such as VLA-4, are required to complete the centripetal transport of SLP-76 microclusters at the IS in a process mediated by contractile Myosin IIA (30). In this regard, SLP76 phosphorylation at pY145 was previously shown to be hampered in SSH1-depleted T cells (10). Beyond actin, which can be regulated by SSH1‐induced cofilin activation (11,13), thereby preventing excessive F-actin rigidity and accumulation at the IS (10), SSH1 activity also impacts microtubule dynamics at the synapse. Interestingly, loss of SSH1 led to increased F-actin at the centrosome area and major tubulin acetylation, impaired centrosome and Golgi polarization to the IS, and lowered microtubule growth when the centrosome reached the IS ( Fig. 6 ). Recently, F-actin at the centrosome area was shown to decrease upon TCR activation, and this is relevant for IS formation, not only in T cells, but also in B cells (46). This is due to the requirement for F-actin clearance at the centrosome area to allow centrosome polarization and directed secretion at the IS (47,48), in a process regulated by PKA (49). This observation is consistent with the increased F-actin accumulation at the IS in SSH1-deficient T cells (10). Golgi complex is also found far from the IS in SSH1-deficient cells, since the reorientation of the Golgi and associated vesicles depends on the correct localization of the centrosome at the IS (50). This is also relevant to sustain long-term TCR activation, thanks to the polarized delivery of signaling molecules at the IS, such as LAT, a relevant scaffold (51). In this context, proper orientation of the centrioles and the centrosome is crucial for the organization of the microtubular network at the IS (32), and centrosome localization at the IS is also supported by tubulin dynamics. This is supported by early HDAC6 activity, promoting deacetylation of pre-existing MTs a few minutes before T cell activation, which coincides with centrosome translocation toward the IS (34), kinesin activity (52) and later increase in MT stabilization after polymerization, since acetylated tubulin at lysine 40 is a marker of microtubule stability (53). Concerning this, SSH1-deficient T cells showed a sustained increase in K40-acetylated-α-tubulin during T cell activation, with a concomitant defect in microtubule growth and dynamics observed by live-cell TIRFm. In addition, Aurora A kinase, which is phosphorylated at T288 and activated early upon TCR activation and regulates Lck activity, helps centrosome translocation toward the IS and MT growth to enable TCR early signaling, intracellular trafficking at the IS (31), as does its downstream effector, Polo-like kinase 1 (Plk1), regulating target cell killing (54). Interestingly, increased Aurora A phosphorylation at T288 augmented T cell signaling in transgenic mice (33), but not in SSH1-deficient CD4 T cells, where exacerbated phosphorylation of Aurora A did not rescue centrosome polarization. This also happens with increased phosphorylation of LIMK1/2 pT508/505 (10) and this study. Indeed, LIMK is involved in actin-microtubule crosstalk through the regulation of Aurora A by phosphorylation (55). These results indicate that SSH1-deficient cells show defects that cannot be rescued by increased activity of kinases, that probably rely on differential positioning of proteins in complexes in a SSH1-dependent manner and warrant further research. Centrosome translocation toward the IS establishes the site of Golgi localization, and both allow the trafficking of vesicles and mitochondria toward the IS (36,56). In T lymphocytes, mitochondria preferentially localize near the IS during T cell activation, in a process regulated by Drp1 through actomyosin-dependent centripetal flux control (36). At the IS, mitochondria regulate calcium signaling (57,58) and orchestrate IS stability through ATP production via oxidative phosphorylation (39), two important factors needed for T cell activation (8). The transport and localization of mitochondria toward the IS not only depend on integrin adhesion (6), but also on tubulin cytoskeleton in collaboration with kinesin and dynein motor proteins (34,58). In addition, CXCL12 chemokine induced inside-out activation of LFA-1 can even pre-polarize the centrosome and mitochondria toward a prospective synapse independently of TCR signals, priming the T cell for robust Ca 2+ influx and NFAT signaling upon antigen recognition (6). Concerning this, SSH1 overexpression in JK T cells leads to permanent activation of cofilin and suppression of CXCL12-mediated directed migration (59). Together, these observations align with our data showing that SSH1-deficient T cells exhibit an impaired activation and localization of LFA-1 integrin, but also an impaired centrosome polarization and Golgi orientation, which could explain the hampered polarization of mitochondria at the IS. Moreover, in migrating T cells, mitochondria redistribution, as well as the centrosome, depends on dynein/dynactin complex in collaboration with Miro-1, an adaptor molecule that couples mitochondria to microtubules (60,61). This is consistent with the increased levels of kinesin and actin observed in isolated mitochondria from TCR-stimulated SSH1 KO JK T cells, which may cause an imbalanced or inhibited transport of these organelles after T cell activation due to altered stoichiometry of motor/regulatory protein complexes. It has been previously described that mitochondrial localization is critical for its function (8,58,62). Although homeostatic ROS production is determinant for TCR derived signaling and NFAT translocation to the nucleus promoting IL-2 production (63), mitochondrial ROS overproduction has been associated with T cell senescence (64) and systemic lupus erythematosus (65). A similar observation has been reported for mitochondrial membrane potential, since mitochondria hyperpolarization, together with an increased mitochondrial ROS production and a defective ATP production, has been reported in T cells from human type 1 diabetes and systemic lupus erythematosus patients (65,66). This evidence supports the altered localization of mitochondria in SSH1-deficient cells as a mechanism to explain the defect in mitochondrial function observed in these cells, with increased ROS production and ΔΨm, resulting in affected OXPHOS. This effect may be due to defects in the Akt/mTOR/S6 signaling pathway, which is involved in glucose-derived metabolism and ribosomal synthesis in T cells (67), in SSH1-silenced T cells, although signaling was still present. Thereby, other mechanisms can be acting in this scenario. In this regard, other metabolic organelles are mobilized together with mitochondria, such as peroxisomes and LDs that polarized to the IS in activated T cells, reflecting a broader organelle orchestration that accompanies IS formation (68). The polarization of mitochondria and peroxisomes to the IS has been proposed as a mechanistic link between the metabolic shifts and the signaling network in T cells, with important implications in cancer immunotherapy (9). Peroxisomes cooperate with mitochondria and LDs to channel FA into oxidative pathways and their relocation in activated T cells may facilitate efficient energy production and redox homeostasis required for proper sustained signaling (69). Concerning this, SSH1-deficient cells fail to polarize peroxisomes to the IS, while their number and interaction with mitochondria are increased together with ROS production in SSH1-deficient CD4 T cells. We propose that SSH1, by linking actin turnover at different locations in cells, and by bridging Talin-1/ADAP complexes and Myosin IIA, creates functional local actin scaffolds necessary to regulate actin dynamics and dynamic microtubule network at different locations, such as F-actin at the IS, at the centrosome area and even at the mitochondria. The resulting actin–microtubule crosstalk ensures the function of molecular motors, such as Myosin IIA and kinesin-1 in the reorientation of organelles such as mitochondria, peroxisomes and LDs and the establishment of relevant contacts to regulate their activity. Fatty acid metabolism is concurrently reprogrammed in T cells, with increased lipid synthesis and enhanced FAO, supporting T cell differentiation and long-term survival (70, 71). Although lipid metabolism and FAO play an important role in the differentiation and maintenance of memory T cells (70, 71), an excessive increase in both lipid accumulation and utilization could alter short-term T cell activation, promoting an exhausted T cell phenotype, characterized by lipotoxicity and lipid peroxidation (72). In this regard, our findings reveal that SSH1 control of the synapse extends to T cell lipid metabolism. SSH1-deficient T cells display excessive lipid droplets accumulation, increased lipid peroxidation, enhanced LD-peroxisome–mitochondria contacts, and elevated FAO, suggesting a shift toward lipid oxidative metabolism. Furthermore, SSH1-silenced T cells showed sustained OCR after injection of etomoxir, an inhibitor of CPT1 transport, which blocks the entry of FA into mitochondria, thereby inhibiting FAO. This suggests that SSH1-silenced cells have a compensatory mechanism, in which junctions between LDs and peroxisomes allow the latter to directly access fatty acids stored in LDs for β-oxidation in peroxisomes. Recent findings reveal that LD trafficking to peroxisomes is essential for maintaining mitochondrial metabolism (73,74), and that the impairment in the crosstalk between mitochondria and endoplasmic reticulum (ER) impedes the use of glucose-derived pyruvate as mitochondrial fuel, causing a shift to FA to sustain energy production (75). In addition, the interaction between LDs, ER and mitochondria helps the recruitment of peroxisomes to the organelle-metabolic hub, supporting FA efflux from LD and enhancing lipid metabolism (76). Regarding this, the increased contacts between these three organelles in SSH1-deficient cells likely create an integrated network of lipid catabolism: fatty acids can be mobilized from LDs and transferred directly to peroxisomes and mitochondria. Overall, increased contacts between LDs, peroxisomes, and mitochondria in SSH1-deficient T cells are expected to enhance FAO, explaining why these cells maintain OCR better even when mitochondrial FA import is pharmacologically blocked with etomoxir. We hypothesize that robust synapse formation via SSH1 couples to mitochondrial and glycolytic glucose fueling of effector functions, whereas loss of SSH1 forces cells to rely more on FAO to meet energy demands. This concept fits emerging paradigms: activated T cells reposition mitochondria to the synapse where they buffer Ca 2+ and supply ATP for actomyosin contractility, while peroxisomes and lipid stores cooperate with mitochondria to sustain redox homeostasis. In summary, our data position SSH1 as a key regulator of T cell activation, enabling TCR-driven actin remodeling that primes LFA-1 for high-affinity adhesion, while simultaneously fostering dynamic microtubules and organelle re-localization to meet the biosynthetic needs of activation. This integrated mechanism, coupling cytoskeletal reorganization to metabolic shift, ensures that signal transduction, adhesion and energy supply progress in concert ( Fig. 6 ). By focusing on the role of SSH1, we provide novel insight on actin dynamics feed into integrin activation and synapse maturation, which may serve to modulate T cell function. Methods Cell lines and human CD4 T cell isolation from peripheral blood The human Jurkat E6-1 CD4 T cell line (Vαl.2 Vβ8 + TCR) and the lymphoblastoid B cell line Raji (Burkitt lymphoma; obtained from the DSMZ Organization; ACC-319) were cultured in RPMI 1640 + GlutaMAX–I + 25 mM HEPES (Gibco–Invitrogen) and supplemented with 10% fetal bovine serum (FBS) (Hyclone, Thermofisher). All lymphoid cell lines were routinely tested for specific expression of CD (clusters of differentiation) with specific antibodies by flow cytometry and for mycoplasma infection by PCR. SSH1 KO cells were generated using CRISPR–Cas9 method as described (10). Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors by separation on a Biocoll gradient (Biochrom) according to standard procedures. CD4 T cells were purified from PBMCs using an EasySep negative isolation kit for human CD4 T cells (17952; Stem Cell Technologies). CD4 T cells were nucleofected with control or SSH1-specific siRNAs (2.5 µM) in Opti-MEM I (Gibco-Invitrogen) and used for activation assays 48 h post-transfection. These studies were performed adhered to the principles of the Declaration of Helsinki and were approved by the local ethics committee for basic research at the ‘ Hospital La Princesa ’. Informed consent was obtained by Centro Transfusiones Comunidad Autonoma de Madrid (CAM). Antibodies, reagents and probes The commercial primary antibodies used in this study were anti-phospho-Aurora A T288 (ab83968; 1:500 for WB), anti-Aurora A (a13824; clone 35C1; 1:500 for WB), anti-GLG1 Golgi complex (ab103439; 1:100 for IF) and anti-GFP (ab13970; 1:200 for IF) from Abcam; anti-ADAP/SLAP-130/Fyb (07-546; 1:500 for WB, 1:50 for IP), anti-β-actin (AM4302; clone AC-15; 1:2,000 for WB), anti-α-tubulin (T6199; clone DM1A; 1:2,000 for WB), anti-(Lys40)-Acetyl-α-tubulin (32-2700; clone 12B4; 1; 1:2,000 for WB, 1:1,000 for IF) and fluorescein isothiocyanate (FITC)-conjugated anti-α-tubulin (F2168; clone DM1A; 1:100 for IF) from Sigma Aldrich; anti-Kinesin Heavy Chain (MAB1613;clone H1; 1:500 WB), anti-Kinesin Light Chain (MAB1617; clone L1; 1:500 for WB) and anti-Dynein Intermediate Chains (MAB1618; clone 74.1; 1:500 for WB) were from Merck Millipore; anti-phospho-LIMK1/2 Y507/T508 (07-850; 1:500 for WB), anti-phospho-Pyk2 Y402 (44-618G; 1:1,000 WB), anti-VDAC (PA1-954A; 1:500 for WB), anti-Pex14 (PA5-78103; 1:100 for IF, 1:100 for FACS), anti-Myosin-IIA (A304-490A; 1:2,000 for WB, 1:200 for IP) and anti-Talin-1 (14168-1-AP; 1:1,000 for WB, 1:100 for IP, 1:100 for IF) were from ThermoFisher-Invitrogen; anti-CD4-PE (317410; clone OKT4; 1:200 for FACS), anti-CD4-APC (317416; clone OKT4; 1:200 for FACS) and anti-CD3ε (clone HIT3a) for stimulatory surfaces were from BioLegend. anti-p150 glued (610474; 1:500 for WB), anti-CD28 (555726; clone CD28.2; 6.67 μg/mL) for stimulatory surfaces and anti-PKCθ (610090; 1:500 for WB) were from BD Pharmingen; anti-Perilipin 2 (GP46; 1:50 for IF) was from Progen; anti-Tom20 (11802-1-AP; 1:2000 for WB, 1:500 for IF) was from Proteintech; anti-phospho-mTOR S2448 (2971S;1:1,000 for WB), anti-mTOR (2972S;1:1,000 for WB), anti-phospho-S6 S235/S236 (2211S;1:1,000 for WB), anti-S6 ribosomal subunit (2217S;1:1,000 for WB), anti-phospho-Akt S473 (9271S;1:1,000 for WB), anti-Akt (9272S;1:1,000 for WB), anti-phospho-MLC2 T18/S19 (3674S;1:1,000 for WB), anti-MLC2 (3672S;1:1,000 for WB), anti-phospho-PKCθ T538 (9377S;1:1,000 for WB), anti-Kindlin-3 (10459S; 1:1,000 for WB, 1:100 for IP, 1:100 for IF), anti-PCM-1 (5259S; 1:100 for IF) and anti-SSH1 (13578S;1:1,000 for WB, 1:100 for IP) were from Cell Signaling Technology; anti-LIMK1 (sc-515585; 1:200 for WB) and anti-SSH1 (sc-517226; 1:20 for IF) were from Santa Cruz Biotechnology. Ghost Dye Red 780 (13-0865; 1:500 for FACS) and Ghost Dye Violet 510 (13-0870; 1:500 for FACS) were from Tonbo Biosciences. The following mAbs: anti-m24 and anti-KIM127 mAbs which recognize the activation-dependent epitopes of LFA-1 integrin and anti-HUTS-21, which recognizes the activation-dependent epitope of VLA-4 integrin, have been described previously (26,27); anti-CD11a (clone TP1/40) and anti-CD49d (clone HP2/1) to detect total LFA-1 (αL) and VLA-4 (α4) integrins, respectively, were produced in our laboratory (77). The anti–phospho-Vav Y174 mAb was a kind gift from Dr X. Bustelo (Centro de Investigación del Cáncer). Cell tracker CMAC (7-amino-4-chloromethylcoumarin; 10 μM ,C2110) was from Molecular Probes, Invitrogen; SEE (0.5 μg/mL, PE404) was from Toxin Technologies; Prolong gold antifade mounting medium (P-36934), prolong gold antifade mounting medium with DAPI (P-36931), Dynabeads M-280 sheep anti-rabbit IgG (11203D) and Dynabeads M-280 sheep anti-mouse IgG (11202D) were from ThermoFisher Scientific; fibronectin and poly-L-Lysine were from Sigma Aldrich; recombinant ICAM-1 was from Hölzel Diagnostika Handels GmbH; anti-CD3/anti-CD28 antibodies (ImmunoCult human T cell activator; 10991) and human recombinant IL-7 (78053) were purchased from Stem Cell Technologies. Reagents and probes were as follows: MitoTracker Orange CMTMRos (M7510; 500 nM for IF), Nonyl-acridine orange (NAO; A1372; 25 nM for FACS) for mitochondrial mass determination, MitoTracker Deep Red FM (M22426, 500 nM for FACS) for mitochondrial membrane potential quantification, MitoSOX Green (M36006; 1 µM for FACS) for ROS determination, BODIPY 581/591 C11 (D3861; 0.8 µM for FACS) for lipid peroxidation detection and BODIPY 493/503 (D3922; 0.8 µM for FACS) for neutral lipids quantification were from Life Technologies-Invitrogen. The following secondary reagents were used: phalloidin conjugated to Alexa Fluor 647 (A-22287; 1:40 for IF), phalloidin conjugated to Alexa Fluor 488 (A-12379; 1:40 for IF), goat anti-rabbit and goat anti-mouse highly cross-adsorbed secondary antibodies conjugated to Alexa Fluor 488 (A-11034 and A-11029, respectively; 1:500 for IF), 568 (A11036 and A-11031, respectively; 1:500 for IF) or 647 (A-21443 and A-21236, respectively; 1:500 for IF), donkey anti-goat highly cross-adsorbed secondary antibody conjugated to Alexa Fluor 647 (A-21447; 1:500 for IF), donkey anti-rabbit secondary antibody conjugated to Alexa Fluor 555 (A-31572; 1:500 for IF), goat anti-chicken antibody conjugated to Alexa Fluor 488 (A-11039; 1:500 for IF) and goat anti-mouse IgG highly cross-adsorbed secondary antibody conjugated to PE (M30004-1; 1:500 for FACS) were purchased from ThermoFisher Scientific; horseradish peroxidase(HRP)-conjugated secondary antibodies for WB (anti-rabbit 31460, anti-mouse 31430 or anti-goat IgG+IgM 31460; all 1:5,000) were purchased from ThermoFisher Scientific. Rabbit TrueBlot ULTRA: anti-rabbit IgG HRP (18-8816-31; 1:1,000) and mouse TrueBlot ULTRA: anti-mouse IgG HRP (18-8817-30; 1:1,000) were from Rockland. Fluorescence-labelled secondary antibodies IRDye 680 goat anti-rabbit and IRDye 800 goat anti-mouse (926-68071 and 926-32350, respectively; 1:5,000 for WB) were from LI-COR Bioscience. Plasmids and siRNA Transfections Double-stranded control (UUCUCCGAACGUGUGCACG and CGUGCACACGUUCGGAGAA) and SSH1-specific (CGGAGAACCUAAACAACAA and UUGUUGUUUAGGUUCUCCG) siRNAs were purchased from Eurogentec. EB3-GFP plasmid was a kind gift from A. Akhmanova (Utrecht University, Netherlands). For Jurkat T cell transfection, cells were centrifuged at 1200 rpm for 5 min, washed with Hank’s balanced salt solution (HBSS, Lonza) and resuspended in Opti-MEM I (Gibco–Invitrogen) (1 × 10 7 cells in 400 μL). 10 µg of plasmids or 2.5 μM siRNAs were added to the cell suspension, which was electroporated in a Gene-Pulse III system (Bio-Rad) set at 240 V, 975 mΩ. After electroporation, cells were resuspended in 5 mL RPMI 1640 + GlutaMAX–I + 25 mM HEPES medium + 5% FBS, plated in 25 cm 2 flasks and supplemented with 10% FBS after 3 h. Experiments were performed 24 h post-transfection. Primary CD4 T cells (5 x 10 6 cells in 100 μL) were nucleofected with control or SSH1-specific siRNAs (2.5 μM) in pre-warmed Opti-MEM I (Gibco-Invitrogen) using the U-04 programme of Nucleofector I (Amaxa) after a heat-shock step with cold HBSS. Cells were cultured for 48 h in RPMI 1640 supplemented with 10% FBS and IL-7 (10 ng/mL). Dead cells were discarded using Biocoll Separating Solution 24 h post-transfection. Experiments were performed 48 h post-transfection; specific silencing was verified by Western blot (Extended Data Fig. 1a). T cell activation and lysis for immunoprecipitation (IP) and immunoblotting For human primary CD4 T cell or Jurkat activation with antibodies, 1 x 10 6 cells per condition in 100 μL of RPMI were stimulated with 20 μL αCD3αCD28 antibodies (ImmunoCult Human CD3/CD28 T-Cell Activator; Stem Cell Technologies) for the indicated times. For JK-Raji conjugate formation, 1 x 10 5 Raji B cells were pulsed with 0.5 μg/mL SEE (1 h, 37ºC, complete medium), washed and mixed with 1x10 6 Jurkat E6-1 T cells (1:10) for the indicated times. Cells were centrifuged at 800 rpm at 37°C to promote conjugation. 1 x 10 6 JK T cells were lysed in 50 μL of 5 mM Tris-HCl pH 7.5 containing 1% NP40, 0.2% Triton X-100, 150 mM NaCl, 2 mM EDTA, 1.5 mM MgCl 2 with phosphatase and protease inhibitors (Phosphostop and Complete tablets from Roche, respectively) for 30 min on ice followed by a preclearance step by centrifugation at 14,000 rpm (4°C, 10 min) to remove debris and nuclei. Samples were processed for SDS-PAGE, transferred to nitrocellulose membranes, blocked with TBS containing 0.2% Tween and 5% BSA and incubated with appropriate primary (o/n, 4°C) and peroxidase-labelled secondary antibodies (1 h, RT). Chemiluminescence was detected using the Amersham 880 detection system (GE Healthcare). For fluorescent Western blot, IRDye 680 goat anti-rabbit and IRDye 800 goat anti-mouse secondary antibodies (Li-Cor Biosciences) were also incubated for 1 h at R/T and detected using the Odyssey Infrared Imager (LI-COR Bioscience). For immunoprecipitation, 1 x 10 7 JK cells per condition were stimulated or not with 50 μL αCD3αCD28 antibodies (ImmunoCult Human CD3/CD28 T-Cell Activator; Stem Cell Technologies) in 1 mL of incomplete RPMI for 10 min, centrifuged and lysed in PHEM buffer (60 mM PIPES, 25 mM HEPES, 5 mM EGTA, 2 mM MgCl 2 ) containing 0.33% Brij 96v supplemented with protease and phosphatase inhibitors for 30 min at RT. Anti‐SSH1 rabbit antibody (10 µg), anti‐Talin-1 rabbit antibody (10 µg), anti-ADAP mouse antibody (10 µg), anti-Myosin IIA rabbit antibody (10 µg) and anti‐Kindlin-3 rabbit antibody (10 µg) were used for immunoprecipitation during 2 h at RT. The complete procedure, including washes and centrifugation, was performed at RT. Preclearing and antibody recovery were performed using 100 μL of dynabeads M-280 sheep anti-rabbit IgG or dynabeads M-280 sheep anti-mouse IgG per IP. Rabbit or mouse serum were used as negative controls (IgG control). Immunoprecipitates and inputs were boiled for 10 min at 85ºC in Laemmli sample buffer 1x containing 5% β-mercaptoethanol and processed for SDS-PAGE. Blots were revealed using True Blot secondary antibodies (1:1,000; Rockland) for detection of primary antibodies and chemiluminescence was detected using the Amersham 880 detection system (GE Healthcare). For the quantification and statistical analysis, immunoprecipitates were normalized by dividing the co-IP prey band intensity by the bait band intensity in the same lane and correcting for the bait which was pulled down. A fold-change was calculated setting unstimulated control value to 1 and dividing all other prey/bait ratios by the control`s ratio (78). Densitometric analysis and quantification of Western blots Bands from Western blots were quantified (arbitrary units per pixel) using either the supplied Image Gauge (Fujifilm Inc) or Image Studio Lite (v5.2, LI-COR Biosciences) software. Background was subtracted and the resulting values were normalized to unstimulated control samples. The data obtained were statistically analyzed and plotted using PRISM8 (GraphPad software). Cell conjugate formation and immunofluorescence experiments For cell conjugate formation (79, 80), Raji B cells (1 x 10 5 cells per coverslip) were washed once with HBSS and loaded with the CMAC cell tracker (10 μM; Molecular Probes) and with SEE (0.5 μg/mL; Toxin Technologies) for 1 h at 37°C in incomplete RPMI. Raji B cells were then washed twice with complete RPMI, conjugated with JK T cells (ratio 1:1) and then attached to poly-L-Lys-coated coverslips for 10 min (for K40-α-tubulin acetylation determination and integrin activation assays), for 15 min (for mitochondrial membrane potential determination using MitoTracker Orange/Tom20 MFI ratio) or for 30 min (for centrosome and organelle polarization assays) at 37°C. For LFA-1 and VLA-4 integrins active conformation detection by IF, m24, KIM127 or HUTS-21 mAbs were added during JK-Raji cell conjugation prior to fixation, to avoid epitope loss during the fixation process. Negative controls were Raji B cells unloaded with SEE and conjugated with JK cells. CD4 T cells were incubated for 10 min (for K40-α-tubulin acetylation determination) or 30 min (for centrosome and organelle polarization assays) at 37°C on coverslips coated with αCD3 (15 µg/mL, clone HIT3a) and αCD28 (5 µg/mL, clone CD28.2) or conjugated with anti-CD3 (20 μg/mL; clone HIT3a) and anti-CD28 (6.67 μg/mL; clone CD28.2) coated latex microbeads (6.4 μm in diameter, Sigma Aldrich) for 30 min at 37°C and were allowed to spread over poly-L-Lys plus fibronectin-coated coverslips. Negative controls were CD4 T cells conjugated with 100 μg/mL human γ-globulin-coated beads or settled over 50 µg/mL fibronectin-coated-coverslips. Cells were then fixed with 4% paraformaldehyde in PHEM (PIPES 30 mM, HEPES 20 mM, EGTA 2 mM, MgCl 2 1 mM, pH: 6.9) containing 0.12 M sucrose for 10 min (R/T), permeabilized with TX-100 (0.2%) in PHEM for 5 min at R/T and blocked with PHEM containing 100 μg/mL γ-globulin, 3% BSA, 0.2% azide for 30 min at R/T. Cells were sequentially stained with the indicated primary antibodies (1-10 μg/mL) followed by Alexa Fluor 488-, 568- or 647-conjugated secondary antibodies (4 μg/mL), Alexa-conjugated phalloidin (5 μg/mL) or FITC-conjugated anti-α-tubulin (0.1 μg/mL). Samples were mounted on Prolong gold or Prolong gold-DAPI (Invitrogen). A series of fluorescence and brightfield images were captured using a TCS SP5 confocal laser scanning unit (Leica Microsystems) attached to an inverted epifluorescence microscope (DMI6000) fitted with an HCX PL APO 63x/1.40-0.6 oil objective or a Leica STELLARIS Navigator confocal microscope equipped with a pulsed WLL (range, 470–670 nm) and an HC PL Apo CS2 100×/1.4 oil objective (Leica Microsystems). Epifluorescence images from CD4 T cells conjugated with latex microbeads were acquired as a Z-series of fluorescence and brightfield images under a THUNDER Imager Live Cell & 3D Cell Culture & 3D Assay and processed with the accompanying thunder algorithm for deconvolution (Leica Microsystems). A 100x objective was used. Images were processed and analyzed using Image J software (http://rsbweb.nih.gov/ij/) and IMARIS software (Bit-plane) (https://imaris.oxinst.com). The ‘Synapse Measures’ plugin (http://rsbweb.nih.gov/ij/) was used to quantify mitochondria, peroxisomes or LD accumulation at the contact area (81). This program provides accurate measurements of localized immunofluorescence by comparing fluorescence signals from multiple regions of the T cell, APC, IS and after subtraction of background fluorescence. Maximal projections and 3D analysis of the T cell-APC contact area were generated using ‘Z-project’, ‘Reslice’, ‘Plot profile’ and ‘3D surface plot’ functions of Image J. Colocalization was measured by using the built-in tool ‘Colocalization threshold’ and representing the Pearson’s or Manders coefficient. The distance of the centrosome or Golgi complex to the IS was calculated using IMARIS software (v8.4) by calculating volumes based on MFI and using the matlab implemented utility ‘spots to volume distance’ (82). Preparation of stimulatory surfaces for TIRFm Glass-bottom-18-well chambers (81817; Ibidi) were coated with 50 µL fibronectin (50 µg/mL) for 3 h at 37°C followed by 50 µL of anti-CD3ε (20 μg/mL; HIT3a clone) and anti-CD28 (6.67 μg/mL; CD28.2 clone) monoclonal antibodies previously diluted in bicarbonate buffer (0.1 M NaHCO 3 and 0.32 M Na 2 CO 3 ) o/n at 4ºC. Before imaging, chambers were washed three times with HBSS, covered with 200 µL of imaging medium (HBSS supplemented with 2% FBS and 25 mM HEPES) and stored at 37°C until use. Live-cell TIRFm acquisition and image analysis of microtubule dynamics For total internal reflection fluorescence microscopy (TIRFm), control and SSH1 KO cells were transfected in a Gene-Pulse III system (Bio-Rad) to overexpress EB3-GFP. Cells were incubated for 24 h, washed, and resuspended in imaging medium (1 x 10 6 cells/100 μl). Then, 20 µL (2 x 10 5 cells) were seeded onto glass-bottom-18-well chamber coated with stimulatory anti-CD3ε and anti-CD28 monoclonal antibodies. Imaging was performed using Leica AM TIRF MC M system mounted on a Leica DMI 6000B fitted with a HCX PL APO 100x1.46 NA oil objective microscope, coupled to an Andor-DU8285 VP-4094 camera. EB3-GFP was excited with the 488 nm laser at 2-5% laser power. Frames were acquired every 300 ms for 3 min and 100-200 ms of exposure time, with a Z penetrance of 150 nm. Synchronization was performed with the accompanying Leica software, and images were analyzed using “ TrackMate ” plugin from ImageJ software (http://rsbweb.nih.gov/ij/). Laplacian of Gaussian (LoG Detector) and Linear Assignment Problem (LAP) tracker were used. To detect the EB3-GFP tips, the following values were used: Estimated object diameter : 0.5 µm. Linking Max distance : 0.5 µm; Gap-closing Max frame gap : 1–2 frames; Gap-closing Max distance : 1.0 µm. Speed, number of tracks and displacement were calculated. TrackMate outputs were post-processed using a Python pipeline to compute per-cell microtubule growth descriptors. Code and a detailed documentation are available in the repository instructions (https://github.com/MLozanoPrieto/trackmate-eb3-tirfm-analysis). Flow cytometry staining 1-3 x 10 5 cells of each cellular type were employed in each flow cytometry staining. Primary and secondary antibody staining was maintained for 30 min on ice and washed with FACS buffer (HBSS, 50 µg/mL human γ-globulin, 2% BSA, 1 mM EDTA). For LFA-1 and VLA-4 integrins active conformation detection by flow cytometry, 3-5 x 10 5 human primary CD4 T cells or JK T cells were activated in 96-well plates coated with αCD3 (20 µg/mL, clone HIT3a) and αCD28 (6.67 µg/mL, clone CD28.2) for 15 min and simultaneously incubated with m24, KIM127 or HUTS-21 mAbs, to detect the active conformation of LFA-1 and VLA-4, respectively. Then, cells were washed three times with HBSS and stained with goat anti-mouse IgG (H+L) secondary antibody conjugated to PE (1:500; ThermoFisher) for 30 min on ice. Finally, cells were washed twice with FACS buffer and resuspended in 200 μL of FACS buffer for flow cytometry acquisition. For peroxisome detection by flow cytometry, control and SSH1 KO JK T cells were fixed with fixation buffer (420801; BioLegend) for 30 min on ice, washed with permeabilization buffer (421002; BioLegend) and incubated with anti-Pex14 rabbit antibody (1:100; ThermoFisher) for 30 min on ice followed by goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (1:500; ThermoFisher) in permeabilization buffer. Finally, cells were washed with permeabilization buffer and resuspended in 200 μL of FACS buffer for flow cytometry acquisition. Data were acquired using a FACS Canto II analyzer cytometer (405 nm violet laser, 488 nm solid state blue laser and 633 nm He-Ne) (BD Biosciences) and analyzed using FlowJo software (v10.7) (BD Biosciences). Isolation of mitochondria for Western blot analysis 1 × 10 7 control or SSH1 KO Jurkat T cells were stimulated or not for 20 min with 50 μL αCD3/αCD28 antibodies (ImmunoCult Human CD3/CD28 T cell Activator) in 1 mL of incomplete RPMI and mitochondria were isolated using a human mitochondria isolation kit following the manufacturer’s instructions (130-094-833; Miltenyi Biotec). Isolated mitochondria were resuspended in 50 μL of RIPA buffer with phosphatase and protease inhibitors (Phosphostop and Complete tablets from Roche, respectively), then sonicated for 1 h and processed for SDS-PAGE in 8 % polyacrylamide gels, transferred to nitrocellulose membranes and subjected to Western blotting. Total lysates were extracted prior to mitochondrial lysis and isolation. Mitochondrial mass, mitochondrial ROS and mitochondrial membrane potential quantification through flow cytometry Mitochondrial mass was assessed in control and SSH1 KO Jurkat or in control or SSH1-silenced human primary CD4 T cells by labeling with nonyl-acridine orange (NAO, 25 nM; Life Technologies-Invitrogen) for 20 min at 37ºC and 5% CO 2 followed by CD4-APC in FACS buffer for 20 min at 4°C. Mitochondrial ROS was determined in control or SSH1-silenced human primary CD4 T cells by labeling with MitoSOX Green (M36006; 1 µM) for 30 min at 37ºC and 5% CO 2 and then stimulated over 96-well plates coated with anti-CD3 (20 μg/mL; clone HIT3a) and anti-CD28 (6.67 μg/mL; clone CD28.2) antibodies for the indicated times. Cells were then stained with Ghost dye Violet 780 Viability Dye (1:1,000, Tonbo Biosciences) in 100 µL of PBS for 30 min at 4°C and CD4-PE for 20 min at 4°C in FACS buffer. Mitochondrial membrane potential was assessed in control or SSH1-silenced human primary CD4 T cells by labeling with MitoTracker Deep Red FM (M22426, 500 nM) for 30 min at 37ºC and 5% CO 2 and then stimulated over 96-well plates coated with anti-CD3 (20 μg/mL; clone HIT3a) and anti-CD28 (6.67 μg/mL; clone CD28.2) antibodies for the indicated times. Cells were then stained with Ghost dye Violet 780 Viability Dye (1:1,000, Tonbo Biosciences) in 100 µL of PBS for 30 min at 4°C and CD4-PE for 20 min at 4°C in FACS buffer. Finally, cells were washed with FACS buffer and resuspended in 100 µL of FACS buffer for flow cytometry acquisition. Mean fluorescence intensity and geometric mean of staining were acquired using a FACS Canto II analyzer cytometer (405 nm violet laser, 488 nm solid state blue laser and 633 nm He-Ne) (BD Biosciences) and analyzed using FlowJo software (v10.7) (BD Biosciences). Real-time cell metabolic analysis using Seahorse The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured using an XF Pro extracellular flux analyzer (Seahorse Bioscience; XFPro M FluxPak Agilent Technologies). For the glucose-based mitostress test, the use of glucose was measured in control and SSH1 KO Jurkat or SSH1-silenced human primary CD4 T cells, in basal conditions or after the stimulation by injection of ImmunoCult Human CD3/CD28 T Cell Activator (Stem Cell Technologies). Cells were cultured with Dulbecco’s modified Eagle medium (DMEM) (D5030, Sigma Aldrich) supplemented with 1 mM sodium pyruvate, 1 mM L-glutamine, and 25 mM glucose and were seeded at 0.3 × 10 6 Jurkat T cells or 0.5 × 10 6 in human primary CD4 T cells per well in culture plates pre-coated with poly-L-Lys (50 µg/mL). Each plate included four independent donors in the case of SSH1-silenced human primary CD4 T cells and five technical replicates. Drugs were injected as follows: oligomycin (1.8 μM), CCCP (2 μM), rotenone (1 μM), and antimycin A (1 μM). Three consecutive mix and measure steps were performed for resting conditions and after each injection (3 min each). For the glycolysis stress assay, cells were cultured with DMEM supplemented with 2 mM L-glutamine and seeded as before (four biological and five technical replicates per plate). Injections were as follows: glucose (10 mM), oligomycin (1.8 μM), and 2-deoxyglucose (2-DG; 50 mM). Mix and measure steps were as before. For the palmitate-based mitostress test, to evaluate the FAO rate, control and SSH1 KO Jurkat or SSH1-silenced human primary CD4 T cells were cultured in substrate-limited growth media (DMEM medium containing 1% FCS, 2.5 mM glucose, 25 mM HEPES, 0.5 mM L-carnitine and 1 mM L-glutamine). The day of the experiment, cells were washed and seeded at 0.3 × 10 6 Jurkat T cells or 0.5 × 10 6 in human primary CD4 T cells per well in FAO assay media (111 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl 2 , 2 mM MgSO 4 , 1.2 mM NaH 2 PO 4 , 1 mM glucose, 0.5 mM L-carnitine, 5 mM HEPES, 125 μM Palmitate-BSA or BSA as control, 40 μM etomoxir or media as control) on poly-L-Lys pre-coated culture plate from XF96 FluxPak (Agilent Technologies). Drugs were injected as follows: medium or ImmunoCult Human CD3/CD28 T Cell Activator, oligomycin (2 μM), CCCP (2 μM), rotenone (1 μM) plus antimycin A (1 μM). Mix and measure steps were as before. Seahorse results were analyzed using Seahorse Wave Pro software. Statistical analysis of time-course OCR/ECAR data was performed in R (v4.2.1) (https://cran.r-project.org) (see Supplementary information). For control and SSH1 KO Jurkat T cells, values were analyzed using linear models (value ~ treatment × condition) followed by estimated marginal means and within-block pairwise contrasts (emmeans; Tukey adjustment). For siCTRL and siSSH1 primary CD4 T cells, data were averaged within each injection block and across technical replicates to obtain one mean per donor × condition × block and analyzed on the log scale with donor as a blocking factor, using HC3 heteroscedasticity-robust standard errors and Holm-adjusted within-block contrasts (emmeans; back-transformed). Lipid droplet and lipid peroxidation quantification through flow cytometry For LD quantification and lipid peroxidation determination, control and SSH1 KO Jurkat T cells were stained with BODIPY 493/503 (0.8 μM; ThermoFisher) or BODIPY 581/591 C11 (0.8 μM; ThermoFisher) in HBSS (Lonza) for 30 min at 37°C in incomplete RPMI, respectively, and LIVE/DEAD Fixable Blue dead cell stain (1:1,000; L23105; ThermoFisher) according to manufacturer's instructions and analyzed by flow cytometry. Lipid peroxidation was obtained as the MFI in the B530/30 channel (peroxidation signal with an emission wavelength of ∼510 nm) of viable cells or the fluorometric ratio between the B530/30 and YG586/15 channels (basal signal with emission wavelength of ∼ 590 nm) as described (83), using a FACS Symphony SORP analyzer (BD Biosciences). Data were analyzed using FlowJo software (v10.7) (BD Biosciences). Statistics and reproducibility Statistical analyses were performed using PRISM8 (GraphPad software). Normality was assessed with Shapiro-Wilk test. Statistical analyses were performed using parametric Student’s t-test, two-tailed paired t-test, and one-way or two-way ANOVA with Bonferroni multiple comparison test, as indicated. Mann-Whitney U and Kruskal-Wallis were used for non-parametric analyses. When comparing two or more samples, including time courses, two-way ANOVA was used. Sample sizes and specific details of each analysis are indicated in the figure legends. Significant differences were considered at p<0.05. Statistical differences are indicated as asterix (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) or non-significant (ns). The schematics and figures were generated with Adobe Illustrator (Adobe, v2020). Declarations Author contributions AGM , conceptualization, experimental design and execution, data curation (cell biology, transfection/nucleofection, flow cytometry, immunoprecipitation assays, Western blot, TIRFm and confocal microscopy), image composition, writing (original draft, review and editing), Fig. 1-5; Supplementary Fig. 1-6; MLP , data curation (TIRFm and Seahorse statistical analysis), writing (review and editing), Fig. 3,4; Supplementary Movie1; CS , CCP , OAS and LMA performed experiments (flow cytometry, microscopy), Fig. 1-2; FSM resources, funding acquisition, data curation, revised manuscript; PRN and NBMC planned and coordinated research, conceptualization, resources, funding acquisition, data curation and interpretation, writing (review and editing). All authors contributed to the article and approved the submitted version. Acknowledgements This study was supported by grants from Madrid Regional Government (S2022/BMD-7209-INTEGRAMUNE-CM) to NBMC, from Spanish Ministry of Science and Innovation funded by MCIN/AEI/10.13039/501100011033 in part granted with ERDF “A way of making Europe” (PID2022-141895OB-I00) to NBMC, (PID2023-147805NB-I00) to PRN, (PID2023-149541OB-I00) to FSM. NBMC and FSM are also funded by Fundación LaCaixa (LCF/PR/HR23/52430018) and CIBER Cardiovascular (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III and co-funded by Fondo Europeo de Desarrollo Regional FEDER). AGM (PIPF-2023/SAL-GL-30092) and CCP (PIPF-2022/SAL-GL-24353) are supported by a PhD Fellowship from the Madrid Regional Government. MLP is supported by an FPI fellowship (PRE2021-097478). CS (PEJ-2021-TL/BMD-21204) and LMA (PEJ-2024-TL/SAL-GL-33552) are supported by “Garantı́a Juvenil'' grant to NBMC from Comunidad de Madrid. OAS is funded by a PhD fellowship of Universidad Complutense de Madrid. Funding agencies have not intervened in the design of the studies, with no copyright over the study. Optical microscopy experimentation was conducted at (1) the Microscopy & Dynamic Imaging, CNIC, ICTS-ReDib, cofunded by MCIN/AEI/10.13039/501100011033 and FEDER Una manera de hacer Europa” (#ICTS-2018-04-CNIC-16) and (2) the Videomicroscopy Facility of the IIS-IP (Madrid, Spain), co-funded by IFEQ21/00085 and IFCS22/00014 from ISCIII and FEDER. We are grateful to Ms. M Ángeles Vallejo and Ana Cayuela for her helpful assistance and management. Competing interests The authors declare no competing interests. Data availability The data underlying this article are available in the article and in its Supplementary Information. Numerical data source is provided in the Supplementary data. All other data and source data are available from the corresponding authors on reasonable request. 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Supplementary Files GomezMoronetalSupplementaryFigures.docx Supplementary Information for Supplementary Figures ExtendedDataFigurelegends.docx GomezMoronetalExtendedDataFig.1.tif Extended Data 1 GomezMoronetalExtendedDataFig.2.tif Extended Data 2 GomezMoronetalExtendedDataFig.3.tif Extended Data 3 GomezMoronetalExtendedDataFig.4.tif Extended Data 4 GomezMoronetalSupplementaryVideo1.avi Microtubule dynamics in CD4 T cells silenced for SSH1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8604514","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":582586734,"identity":"66d27b13-2cd1-4f44-8a31-c79790c366c7","order_by":0,"name":"Noa 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18:51:06","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8604514/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8604514/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106963390,"identity":"d4f58730-4c27-4e69-b255-944ea0c5fd31","added_by":"auto","created_at":"2026-04-15 09:44:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":49994405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSSH1 nucleates Talin-1, Kindlin-3, ADAP and Myosin IIA at the IS\u003c/strong\u003e.\u003cstrong\u003e a \u003c/strong\u003eConfocal fluorescence images showing the localization of SSH1 (magenta), Talin-1 (cyan) and F-actin (green) at the IS in conjugates of JK T cells and CMAC-labelled, SEE-preloaded Raji B cells. Bar, 5 µm. A 3D reconstruction of the IS and a fluorescence intensity profile of SSH1, Talin-1 and F-actin at the IS is shown. \u003cstrong\u003eb-d \u003c/strong\u003eConfocal fluorescence images showing the localization of \u003cstrong\u003eb\u003c/strong\u003e Talin-1 (magenta), \u003cstrong\u003ec\u003c/strong\u003e Kindlin-3 (magenta) and \u003cstrong\u003ed\u003c/strong\u003e ADAP (magenta) and SSH1 (green) at the IS established by human primary CD4 T cells conjugated with anti-CD3/anti-CD28-coated beads. Bar, 5 µm. \u003cstrong\u003ee\u003c/strong\u003e Graphs, Pearson’s co-localization coefficients obtained in individual cell interactions (orange dots). \u003cstrong\u003ef-m \u003c/strong\u003eCo‐immunoprecipitation of SSH1, Talin-1, Kindlin-3, ADAP and Myosin II A in JK E6-1 WT T cells \u003cstrong\u003ef,k\u003c/strong\u003e or in JK \u003cem\u003eSSH1\u003c/em\u003e KO T cells\u003cstrong\u003e l,m\u003c/strong\u003e. Inputs and negative controls are shown. Cells were stimulated (+) or not (-) with anti‐CD3+anti‐CD28 antibodies for 10 min. A representative experiment out of three and the quantification of prey normalized to the unstimulated bait \u003cstrong\u003eg,i,k,m\u003c/strong\u003e are shown. \u003cstrong\u003en\u003c/strong\u003e Scheme showing the interactions regulated by SSH1.\u003cstrong\u003e o\u003c/strong\u003e Western blot showing the phosphorylation of pT18/S19 MLC2 in siCTRL and siSSH1 transfected CD4 T cells after the stimulation with anti‐CD3+anti‐CD28 antibodies for the indicated times in minutes (min). SSH1 and p150\u003csup\u003eglued\u003c/sup\u003e are also shown. A representative experiment out of four is shown. \u003cstrong\u003ep \u003c/strong\u003eGraph showing the ratio of pT18/S19 MLC2 phosphorylation to total protein normalized to unstimulated control. Data in graphs are individual cells (represented as dots) and lines represent the mean ± SD: (e) n=40 cells analyzed from four independent donors; (p) n=4 independent donors, two-way ANOVA. ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"GomezMoronetalFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/dc8f33c40364969fe776080e.png"},{"id":106962135,"identity":"10aa3496-d91a-4325-9be6-338aa8f37d96","added_by":"auto","created_at":"2026-04-15 09:34:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38917410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSSH1 regulates LFA-1 active conformation\u003c/strong\u003e.\u003cstrong\u003e a\u003c/strong\u003e Scheme showing the different conformations of LFA-1 integrin upon TCR stimulation and the antibodies used to detect them. \u003cstrong\u003eb-c \u003c/strong\u003eGraphs showing the geometric mean of \u003cstrong\u003eb\u003c/strong\u003e KIM127 and \u003cstrong\u003ec\u003c/strong\u003e m24 antibodies in siCTRL and siSSH1 transfected CD4 T cells, activated (CD3/CD28) or not (-) over surfaces coated with stimulatory anti-CD3/anti-CD28 antibodies. \u003cstrong\u003ed, f \u003c/strong\u003eConfocal fluorescence images showing the localization of \u003cstrong\u003ed\u003c/strong\u003e KIM127 (cyan) or \u003cstrong\u003ef \u003c/strong\u003em24 (cyan) with LFA-1 (magenta) and F-actin (green) at the IS in conjugates of control or \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells and CMAC-labelled, SEE-preloaded Raji B cells. Bar, 5 µm. \u003cstrong\u003ee, g \u003c/strong\u003eA 3D reconstruction of the IS from (d) and (f) is shown. \u003cstrong\u003eh \u003c/strong\u003eWestern blot showing the phosphorylation of pY402 Pyk2 and pY174 Vav1 in siCTRL and siSSH1 transfected CD4 T cells after the stimulation with anti‐CD3+anti‐CD28 antibodies for the indicated times. SSH1 and p150\u003csup\u003eglued\u003c/sup\u003e are also shown. A representative experiment out of four is shown. \u003cstrong\u003ei\u003c/strong\u003e Graphs, ratio of pY402 Pyk2 and pY174 Vav1 phosphorylation to total protein normalized to unstimulated control. Data are individual experiments shown as dots and lines representing the mean ± SD: (b-c) n=6 independent donors, two-tailed paired t-test, (i) n=4 independent donors, two-way ANOVA. ns, not significant; *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001. See also Extended Data Fig. 1.\u003c/p\u003e","description":"","filename":"GomezMoronetalFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/1e13894275bc986b40533b89.png"},{"id":106963358,"identity":"6078772a-aa21-4c02-b365-0964c36ff6c6","added_by":"auto","created_at":"2026-04-15 09:43:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63894267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSSH1 promotes tubulin cytoskeleton dynamics after TCR activation\u003c/strong\u003e.\u003cstrong\u003e a\u003c/strong\u003eConfocal fluorescence images showing the localization of PCM1 (magenta) and F-actin (green) at the IS in siCTRL and siSSH1 transfected human primary CD4 T cells conjugated with anti-CD3/anti-CD28-coated beads to establish IS. Bar, 5 µm. \u003cstrong\u003eb\u003c/strong\u003e Graph showing the centrosome-IS distance from (a). \u003cstrong\u003ec \u003c/strong\u003eConfocal fluorescence images showing the localization of PCM1 (green in the merged) and F-actin (magenta in the merged) at the IS in conjugates of control or \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells and CMAC-labelled, SEE-preloaded Raji B cells. Bar, 5 µm. Insets 1-4 show magnified areas of the MTOC area (white squares). \u003cstrong\u003ed,e \u003c/strong\u003eGraphs showing\u003cstrong\u003e d \u003c/strong\u003ecentrosome polarization index and\u003cstrong\u003e e \u003c/strong\u003ecentrosomal area F-actin MFI ratio from (c). \u003cstrong\u003ef,g\u003c/strong\u003eGraphs data from (c) showing the correlation between centrosome polarization index and centrosomal area F-actin MFI ratio in f\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eSSH1\u003c/em\u003e KO and \u003cstrong\u003eg\u003c/strong\u003econtrol JK T cells. \u003cstrong\u003eh\u003c/strong\u003e Confocal fluorescence images of siCTRL and siSSH1 transfected CD4 T cell stimulated over surfaces coated with stimulatory anti-CD3/anti-CD28 antibodies. PCM1 (magenta), F-actin (green) and α-tubulin (cyan) are shown. 3D surface plots are shown. \u003cstrong\u003ei\u003c/strong\u003e Graph data from (h) showing the centrosomal area F-actin MFI ratio. \u003cstrong\u003ej\u003c/strong\u003e Confocal fluorescence images of siCTRL and siSSH1 transfected CD4 T cell stimulated over surfaces coated with stimulatory anti-CD3/anti-CD28 antibodies. Golgi (magenta), F-actin (cyan) and α-tubulin (green) are shown. 3D surface plots are shown. \u003cstrong\u003ek \u003c/strong\u003eWestern blot showing the phosphorylation of pT508/505 LIMK1/2 and pT288 Aurora A in control or \u003cem\u003eSSH1\u003c/em\u003eKO JK T cells after conjugation with SEE-preloaded Raji cells for the indicated times. A representative experiment from four is shown.\u003cstrong\u003e l\u003c/strong\u003e Graphs showing the ratio of pT508/505 LIMK1/2 and pT288 Aurora A phosphorylation to total protein normalized to unstimulated control. \u003cstrong\u003em\u003c/strong\u003e Time-lapse TIRFm images from representative control and \u003cem\u003eSSH1\u003c/em\u003e KO cells expressing EB3-GFP (pseudocolour) and stimulated over surfaces coated with stimulatory anti-CD3/anti-CD28 antibodies. Bar, 5 μm.\u003cstrong\u003e \u003c/strong\u003eMaximal projection of the time lapses and brightfield (BF) are also shown. \u003cstrong\u003en\u003c/strong\u003e Graph, speed parameter (µm/s) calculated from (m). \u003cstrong\u003eo\u003c/strong\u003e Western blot showing the acetylation of K40-α-tubulin in control or \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells after conjugation with SEE-preloaded Raji cells for the indicated time. A representative experiment from four is shown. Graph, ratio of K40-α-tubulin acetylation to total protein normalized to unstimulated control. Data are individual experiments or cells shown as dots and lines representing the mean ± SD: (b) siCTRL(-) n=30, siCTRL(+) n=35, siSSH1(-) n=28, siSSH1(+) n=35 cells analyzed from four independent donors, Mann-Whitney test; (d) Ctrl(-) n=41, Ctrl(+) n=88, \u003cem\u003eSSH1\u003c/em\u003eKO(-) n=42, \u003cem\u003eSSH1\u003c/em\u003e KO(+) n=73 cells analyzed from four independent experiments. Mann-Whitney test; (e) Ctrl(-) n=73, Ctrl(+) n=87, \u003cem\u003eSSH1\u003c/em\u003eKO(-) n=75, \u003cem\u003eSSH1\u003c/em\u003e KO(+) n=55 cells analyzed from four independent experiments, Mann-Whitney test; (f) n=87; (g) n=55 cells analyzed from four independent experiments, linear regression of correlation; (i) siCTRL(-) n=58, siCTRL(+) n=50, siSSH1(-) n=64, siSSH1(+) n=50 cells analyzed from four independent donors. Mann-Whitney test; (l) n=4 independent experiments, two-way ANOVA; (n) Ctrl (n=19), \u003cem\u003eSSH1\u003c/em\u003e KO (n=17) cells analyzed from three independent experiments, Mann-Whitney test; (o) n=4 independent experiments, two-way ANOVA. ns, not significant; *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001. See also Extended Data Fig. 2.\u003c/p\u003e","description":"","filename":"GomezMoronetalFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/e80d3bd5f27a98b7a30889a5.png"},{"id":106963450,"identity":"18dd5a03-d105-4ac5-b7cd-2d1a9f03476e","added_by":"auto","created_at":"2026-04-15 09:44:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":42779421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial localization and function and glucose-derived metabolism are regulated by SSH1\u003c/strong\u003e.\u003cstrong\u003e a \u003c/strong\u003eConfocal fluorescence images showing the localization of mitochondria (magenta), LFA-1 (cyan) and F-actin (green) at the IS in conjugates of control or \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells and CMAC-labelled, SEE-preloaded Raji B cells. Bar, 5 µm. \u003cstrong\u003eb \u003c/strong\u003eGraph, data from (a) showing mitochondria polarization to the IS. \u003cstrong\u003ec \u003c/strong\u003eConfocal fluorescence images showing the localization of mitochondria (magenta) and F-actin (green) at the IS in siCTRL and siSSH1 transfected human primary CD4 T cells conjugated with anti-CD3/anti-CD28-coated beads to establish IS. Bar, 5 µm. \u003cstrong\u003ed\u003c/strong\u003e Graph, data from (c) showing mitochondria polarization to the IS. \u003cstrong\u003ee-f\u003c/strong\u003eWestern blotting showing the distribution of \u003cstrong\u003ee \u003c/strong\u003epT508/505 LIMK1/2 and total LIMK1, K40-acetylated and total α-tubulin and β-actin cytoskeletal components or \u003cstrong\u003ef\u003c/strong\u003e kinesin-1 [Kinesin heavy chain (KHC); kinesin light chain (KLC)] and dynein (p74-dynein intermediate chain) molecular motors in isolated mitochondria from control or \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells stimulated or not with anti‐CD3ε+anti‐CD28 antibodies for 20 min. VDAC and Tom20 were loading controls. A representative experiment out of four is shown. \u003cstrong\u003eg-i\u003c/strong\u003e Graphs showing the fold-change in geometric mean from \u003cstrong\u003eg\u003c/strong\u003e NAO (mitochondrial mass), \u003cstrong\u003eh\u003c/strong\u003e mitoSOX Green (ROS) and \u003cstrong\u003ei\u003c/strong\u003e MitoTracker Deep Red (mitochondrial membrane potential) in siCTRL and siSSH1 transfected CD4 T cells, resting or activated for the indicated times over surfaces coated with stimulatory anti-CD3/anti-CD28 monoclonal antibodies. \u003cstrong\u003ej-k\u003c/strong\u003e Fluorescence images of siCTRL and siSSH1 transfected CD4 T cells \u003cstrong\u003ej\u003c/strong\u003e unstimulated or \u003cstrong\u003ek\u003c/strong\u003estimulated over surfaces coated with stimulatory anti-CD3/anti-CD28 monoclonal antibodies. MitoTracker Orange, magenta; Tom20, green. Bar, 5 μm. \u003cstrong\u003el\u003c/strong\u003eGraph showing quantification of the ratio between MitoTracker Orange and Tom20 MFI. \u003cstrong\u003em\u003c/strong\u003e Graphs showing basal respiration, ATP production, maximal respiration and spare respiratory capacity from a glucose-based mitostress test in resting and stimulated siCTRL and siSSH1 transfected CD4 T cells. \u003cstrong\u003en \u003c/strong\u003eGraphs showing the glycolysis and glycolytic capacity from a glucose-based glycostress test in resting and stimulated siCTRL and siSSH1 transfected CD4 T cells. \u003cstrong\u003eo\u003c/strong\u003eWestern blot showing the phosphorylation of pS473 Akt, pS2448 mTOR and pS235/236 S6 ribosomal subunit in siCTRL and siSSH1 transfected CD4 T cells after the stimulation with anti‐CD3ε+anti‐CD28 antibodies for the indicated times. A representative experiment from four is shown. \u003cstrong\u003ep\u003c/strong\u003e Graphs, data from (o) showing the ratio pS473 Akt, pS2448 mTOR and pS235/236 S6 ribosomal subunit phosphorylation to total protein normalized to unstimulated control. Data are individual experiments or cells shown as dots and lines representing the mean ± SD: (b) Ctrl(-) n=66, Ctrl(+) n=83, SSH1 KO(-) n=77, SSH1 KO(+) n=87 cells analyzed from four independent experiments, Mann-Whitney test; (d) siCTRL(-) n=35, siCTRL(+) n=55, siSSH1(-) n=35, siSSH1(+) n=55 cells analyzed from four independent donors. Mann-Whitney test; (g) n=5 independent donors, two-tailed paired t-test; (h) n=5 independent donors, two-way ANOVA; (i) n=7 independent donors, two-way ANOVA; (l) siCTRL(-) n=60, siCTRL(+) n=50, siSSH1(-) n=64, siSSH1(+) n=59 cells analyzed from four independent donors. Mann-Whitney test; (m,n) n=8 independent donors, two-way ANOVA; (p) n=4 independent donors, two-way ANOVA. ns, not significant; *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0. 0001. See also Extended Data Fig. 3.\u003c/p\u003e","description":"","filename":"GomezMoronetalFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/94f7b64a5d64746b4c255218.png"},{"id":106962125,"identity":"2c0fc9d6-2dd3-4032-bc57-22639fbd5e81","added_by":"auto","created_at":"2026-04-15 09:34:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":34564957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSSH1 controls organelle contact at the IS and its absence enhances lipid metabolism in T cells\u003c/strong\u003e.\u003cstrong\u003e a \u003c/strong\u003eConfocal fluorescence images showing the localization of mitochondria (magenta), peroxisomes (cyan) and α-tubulin (green) at the IS in siCTRL and siSSH1 transfected human primary CD4 T cells conjugated with anti-CD3/anti-CD28-coated beads to establish IS. Bar, 5 µm. \u003cstrong\u003eb-c \u003c/strong\u003eGraphs, \u003cstrong\u003eb\u003c/strong\u003e primary CD4 T cells or \u003cstrong\u003ec\u003c/strong\u003e JK peroxisomes polarization to the IS. \u003cstrong\u003ed\u003c/strong\u003e Fluorescence images of siCTRL and siSSH1 transfected CD4 T cells activated over surfaces coated with stimulatory anti-CD3/anti-CD28 monoclonal antibodies. Mitochondria, magenta; peroxisomes, green; α-tubulin, cyan. Bar, 5 μm. \u003cstrong\u003ee-f \u003c/strong\u003eGraphs showing the \u003cstrong\u003ee\u003c/strong\u003e number of peroxisomes per cell and \u003cstrong\u003ef\u003c/strong\u003emitochondria-peroxisome Manders co-localization coefficient from (d). \u003cstrong\u003eg-i\u003c/strong\u003eGraphs showing the geometric mean from \u003cstrong\u003eg\u003c/strong\u003e Pex14 (peroxisomes), \u003cstrong\u003eh \u003c/strong\u003eBodipy 493/503 (neutral lipids) and \u003cstrong\u003ei\u003c/strong\u003e Bodipy 581/591 C11 (lipid peroxidation) in control or \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells. \u003cstrong\u003ej\u003c/strong\u003e Fluorescence images of siCTRL and siSSH1 transfected CD4 T cells activated over surfaces coated with stimulatory anti-CD3/anti-CD28 monoclonal antibodies. Mitochondria, magenta; peroxisomes, green; lipid droplets (LD), cyan. Bar, 5 μm.\u003cstrong\u003e k\u003c/strong\u003e Graph, data from (j) showing the number of LDs per cell. \u003cstrong\u003el,m \u003c/strong\u003eGraphs, data from (j) showing \u003cstrong\u003el\u003c/strong\u003e peroxisome-LD and \u003cstrong\u003em\u003c/strong\u003e mitochondria-LD Manders co-localization coefficient. \u003cstrong\u003en,o\u003c/strong\u003e Graphs showing the mitochondrial oxygen consumption rate (OCR) of \u003cstrong\u003en\u003c/strong\u003e endogenous FAO and \u003cstrong\u003eo\u003c/strong\u003e exogenous palmitate oxidation from a palmitate-based mitostress test in resting and stimulated siCTRL and siSSH1 transfected CD4 T cells. Data are individual experiments shown as dots and lines representing the mean ± SD: (b) siCTRL(-) n=20, siCTRL(+) n=35, siSSH1(-) n=22, siSSH1(+) n=40 cells analyzed from four independent donors, Mann-Whitney test; (c) Ctrl(-) n=41, Ctrl(+) n=70, \u003cem\u003eSSH1\u003c/em\u003e KO(-) n=42, \u003cem\u003eSSH1\u003c/em\u003eKO(+) n=70 cells analyzed from four independent experiments. Mann-Whitney test; (e) siCTRL n=147, siSSH1 n=145 cells analyzed from four independent donors, Mann-Whitney test; (f) siCTRL n=50, siSSH1 n=50 cells analyzed from four independent donors, Mann-Whitney test. (g) n=14 independent experiments; (h) n=5 independent experiments; (i) n=4 independent experiments, two-tailed paired t-test. (k-m) siCTRL n=50, siSSH1 n=55 cells analyzed from four independent donors, Mann-Whitney test; (n) n=8 independent donors, two-way ANOVA; (o) n=5 independent donors, two-way ANOVA. ns, not significant; *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001. See also Extended Data Fig. 4.\u003c/p\u003e","description":"","filename":"GomezMoronetalFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/35effa0aff2f30363a3ba2f7.png"},{"id":106963091,"identity":"35947087-225a-424c-aa56-d0a50a1372fe","added_by":"auto","created_at":"2026-04-15 09:42:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":19429960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic model for the role of SSH1 in coordinating actin and microtubule remodeling to promote metabolic reprogramming after T cell activation. \u003c/strong\u003eLeft panel: SSH1 links Talin-1, Kindlin-3, ADAP, and Myosin-IIA to F-actin, facilitating LFA-1 activation. This connection enables SSH1 to organize F-actin at the centrosomal area, thereby promoting organelle relocalization after T cell activation, including the centrosome and metabolic organelles, such as mitochondria, peroxisomes and lipid droplets. Right panel: Loss of SSH1 disrupts actin organization, leading to reduced LFA-1 activation, defective microtubule dynamics and impaired organelle polarization and organization at the immunological synapse (IS). Created with BioRender.com.\u003c/p\u003e","description":"","filename":"GomezMoronetalFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/b6200b67e18cb1c3260dab12.png"},{"id":106876201,"identity":"bbe5cf2d-b495-47da-9109-32cd668f4ec7","added_by":"auto","created_at":"2026-04-14 10:32:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1333220,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/1408ee87-2626-41ba-a506-9de2ea38dfd7.pdf"},{"id":106963347,"identity":"6c1a1624-81c7-467e-b67c-38af0a25aeab","added_by":"auto","created_at":"2026-04-15 09:43:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1905300,"visible":true,"origin":"","legend":"Supplementary Information for Supplementary Figures","description":"","filename":"GomezMoronetalSupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/b65a66cb763c5194d51c1508.docx"},{"id":106963198,"identity":"28370dbb-7335-4ab5-ae17-6752391c577b","added_by":"auto","created_at":"2026-04-15 09:42:50","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18469,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFigurelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/fb91a5d545a759512989b5b9.docx"},{"id":106962090,"identity":"4036ecb5-52eb-491b-b135-2b38e7a1cb6f","added_by":"auto","created_at":"2026-04-15 09:33:32","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18263284,"visible":true,"origin":"","legend":"Extended Data 1","description":"","filename":"GomezMoronetalExtendedDataFig.1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/feaf237692e8e02c783e4682.tif"},{"id":106963186,"identity":"9444b4da-29c0-47cc-88b6-5e7ba10e4479","added_by":"auto","created_at":"2026-04-15 09:42:46","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14978496,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data 2\u003c/p\u003e","description":"","filename":"GomezMoronetalExtendedDataFig.2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/148aae4f31f2a37c9194b9d2.tif"},{"id":106964903,"identity":"e6290168-92ad-41d3-a807-b47077aa03b2","added_by":"auto","created_at":"2026-04-15 09:52:20","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16706328,"visible":true,"origin":"","legend":"Extended Data 3","description":"","filename":"GomezMoronetalExtendedDataFig.3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/f6f4d2312f1564242c4e8388.tif"},{"id":106963365,"identity":"8cdbba97-0fcb-4fdd-96a5-266cfa29c100","added_by":"auto","created_at":"2026-04-15 09:43:55","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":22893252,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data 4\u003c/p\u003e","description":"","filename":"GomezMoronetalExtendedDataFig.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/d2ab467bcb010ab13d570572.tif"},{"id":106962087,"identity":"0ef00a96-defd-4b56-b46a-a0fa3d595a34","added_by":"auto","created_at":"2026-04-15 09:33:22","extension":"avi","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":20898432,"visible":true,"origin":"","legend":"\u003cp\u003eMicrotubule dynamics in CD4 T cells silenced for SSH1\u003c/p\u003e","description":"","filename":"GomezMoronetalSupplementaryVideo1.avi","url":"https://assets-eu.researchsquare.com/files/rs-8604514/v1/06e669124c8699ca3dadc2d7.avi"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Slingshot-1 (SSH1) phosphatase Controls Cytoskeletal Remodeling, Integrin conformation and Metabolic Reprogramming During CD4 T Cell Activation","fulltext":[{"header":"Main","content":"\u003cp\u003eT cell activation is a tightly coordinated process that couples cell signaling with dynamic cytoskeletal remodeling. Upon T cell receptor (TCR) engagement by antigen, substantial rearrangements of both the actin and tubulin networks occur at the nascent immunological synapse (IS) (1). These cytoskeletal dynamics are essential for sustaining TCR signaling and driving full T cell effector function (2). A critical outcome of these early cytoskeletal events is the complete activation of the integrin LFA-1 to support cell adhesion to the antigen-presenting cell (APC), a process that requires active actin remodeling at the synapse\u0026nbsp;as well as the interaction of Talin-1, Kindlin-3 and ADAP cytoskeletal adaptors (3,4). Beyond adhesion, LFA-1 engagement triggers broader cellular reprogramming at the IS. LFA-1 ligation provides mechanical and co-stimulatory signals that drive the polarization of intracellular organelles toward the IS. For instance, LFA-1\u0026ndash;Erk1/2 signaling promotes centrosome translocation to the IS (5), and LFA-1 activation is required for repositioning of mitochondria to the T cell\u0026ndash;APC interface (6). At the IS, mitochondria serve as localized powerhouses and calcium buffers (7), sustaining the high ATP demands and prolonged Ca\u003csup\u003e2+\u003c/sup\u003e signaling needed for full T cell activation (8). In this manner, the convergence of cytoskeletal regulation and organelle positioning has profound consequences for T cell metabolism and cancer immunotherapy (9).\u003c/p\u003e\n\u003cp\u003eSlingshot-1 (SSH1) phosphatase has emerged as a molecular regulator linking TCR signals to actin cytoskeleton remodeling (10). SSH1 is a dual-specificity phosphatase that dephosphorylates and activates cofilin, thereby promoting actin filament turnover (11,\u0026nbsp;12), and can concomitantly inactivate LIM-kinase (LIMK) to boost cofilin activity (13). SSH1 depletion in CD4 T cells affects CD3\u0026epsilon; conformational change and Nck recruitment, which leads to increased actin dynamics and unstable IS, showing disrupted TCR organization and early signaling in a process regulated by LIMK. (10). Nevertheless, it remains unclear whether these actions on actin organization exert other effects on T cell biology through IS regulation. In this regard, TCR and co-stimulatory signals trigger a well-characterized metabolic rewiring in T cells. Quiescent T cells shift to a highly glycolytic state while also boosting mitochondrial oxidative capacity, thereby meeting the increased energetic and biosynthetic demands of clonal expansion, differentiation and effector function (14,\u0026nbsp;15). Here, we show that SSH1 orchestrates the remodeling of both actin and microtubule networks to ensure optimal CD4\u003csup\u003e+\u003c/sup\u003e T cell activation and metabolic reprogramming. Mechanistically, SSH1 is required for the molecular association of ADAP and Myosin-IIA in a multiprotein complex containing Talin-1 and Kindlin-3 that enables LFA-1 to achieve its fully active conformation and properly localize at the IS. This allows SSH1 to regulate the repositioning of mitochondria, peroxisomes, and lipid droplets at the IS through coordinated actin\u0026ndash;microtubule crosstalk. Consequently, SSH1-deficient CD4\u003csup\u003e+\u003c/sup\u003e T cells show dysregulated mitochondria and peroxisomes activities, including marked ROS production and lipid peroxidation upon IS formation. Collectively, our findings establish SSH1 role in linking adhesion and cytoskeletal dynamics to organelle positioning to facilitate metabolic remodeling and to drive efficient CD4\u003csup\u003e+\u003c/sup\u003e T cell antigenic responses.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSSH1 nucleates Talin-1, Kindlin-3, ADAP and Myosin IIA at the IS \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSSH1 is activated by TCR triggering and regulates actin cytoskeleton remodeling by its phosphatase activity on cofilin and LIMK, with SSH1-deficient cells showing increased actin dynamics and capacity to extend motile lamellae (10). To address the way SSH1 localizes at the IS and how it may propagate the TCR signaling, we first searched for potential interactors between SSH1 and key actin regulators at the IS through confocal microscopy. JK T cells were conjugated with SEE-loaded Raji B cells, acting as APCs to establish the IS. SSH1 co-distributed at these ISs with F-actin and Talin-1 \u003cstrong\u003e(Fig. 1a)\u003c/strong\u003e. The profiles for the distribution of the mean fluorescence intensity (MFI) of these components in 3D-IS modeling revealed that SSH1 accumulated mainly at the external area of the IS, with a similar pattern to Talin-1 and F-actin \u003cstrong\u003e(Fig. 1a)\u003c/strong\u003e. Talin-1 connects actin dynamics to LFA-1 integrin (16), which may serve to help SSH1 localization at the IS and the motile lamellae observed in T cells expressing low or no SSH1 (17). Next, human primary CD4 T cells forming synapse-like structures with beads coated with stimulating anti-CD3 and anti-CD28 antibodies were studied. SSH1 co-localized with Talin-1 (\u003cstrong\u003eFig. 1b,e\u003c/strong\u003e), Kindlin-3 (\u003cstrong\u003eFig. 1c,e\u003c/strong\u003e) and ADAP/Fyb (\u003cstrong\u003eFig. 1d,e\u003c/strong\u003e) at the T cell side of the contact. The molecular association between SSH1, Talin-1, Kindlin-3 and ADAP was confirmed through co-immunoprecipitation (co-IP) assays in resting and stimulated JK T cells \u003cstrong\u003e(Fig. 1f,g)\u003c/strong\u003e. TCR triggering enhanced the interaction of SSH1 with these proteins. Talin-1 and kindlin-3 mainly interacted with each other upon TCR activation and with the other members of the complex. ADAP was the member of the complex that showed more restrictive interaction in resting conditions and showed inducible capacity to co-IP with the other members of the complex upon TCR activation. \u003c/p\u003e\n\u003cp\u003eThese proteins are involved in the complete activation of LFA-1 integrin by propagating TCR signals to the integrin activation machinery (18-20). Indeed, Talin-1 and Kindlin-3 complex regulates the action of Myosin IIA (21), which is required to organize mechanical forces at the IS (22). Therefore, the presence of Myosin-IIA was probed in SSH1 complexes, finding that their association was highly increased upon TCR stimulation (\u003cstrong\u003eFig. 1h,i\u003c/strong\u003e). Indeed, protein-protein associations were observed for Myosin IIA, Talin-1, Kindlin-3 and ADAP upon TCR triggering and co-stimulation (\u003cstrong\u003eFig. 1j\u003c/strong\u003e), with ADAP and especially Talin-1 showing major recovery of the complex upon activation (\u003cstrong\u003eFig. 1j,k\u003c/strong\u003e). When Myosin IIA was immunoprecipitated, it apparently associated with the other proteins comparably in resting and activated cells (\u003cstrong\u003eFig. 1j,k\u003c/strong\u003e). To investigate whether this protein network depended on SSH1, similar experiments were performed in cells lacking SSH1 (\u003cem\u003eSSH1\u003c/em\u003e KO CRISPR/Cas9 JK T cells). In the absence of SSH1, ADAP was unable to properly incorporate in the protein complex, with low or no recovery of Talin-1, Kindlin-3 and Myosin-IIA (\u003cstrong\u003eFig. 1l,m\u003c/strong\u003e). ADAP was completely absent from the protein network when Talin, Kindlin-3 or Myosin IIA were immunoprecipitated.However, the interaction between Talin-1 and Kindlin-3 was conserved, although Myosin IIA was not recovered anymore in the complex (\u003cstrong\u003eFig. 1l,m\u003c/strong\u003e). In contrast, Myosin IIA seemed able to recover Kindlin-3 and Talin-1 irrespective of stimulation in the absence of SSH1 (\u003cstrong\u003eFig. 1l,m\u003c/strong\u003e). These results suggest a potential complex where SSH1 would help Talin-1/Kindlin-3 complex to connect with ADAP and Myosin-IIA, and that this complex could be induced upon TCR activation and formation of the IS. Therefore, SSH1 seems to bring together active molecules to the F-actin structures to regulate cell adhesion (\u003cstrong\u003eFig. 1n\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn support of this, the phosphorylation of the regulatory myosin light chain (MLC) at pT18 and pS19 was decreased in siSSH1 CD4 T cells (\u003cstrong\u003eFig. 1o,p\u003c/strong\u003e). This phosphorylation is a key event for Myosin IIA activation and tension generation (23), which points to SSH1 as potential regulator of Myosin-IIA activity at the IS. SSH1 may therefore serve as a scaffold to recruit connectors of F-actin and membrane receptors, as can be integrins, to help the stability of T-APCs contacts. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eLFA-1 requires SSH1 to adopt its active conformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo gain insight into the potential role of a SSH1-dependent complex including proteins that regulate the activation of LFA-1 integrin (24,25), we investigated SSH1 requirement for specific active, conformational state of LFA-1 (\u003cstrong\u003eFig. 2a\u003c/strong\u003e). Specific extended-closed and extended-open conformational epitopes on LFA-1 were detected through two specific monoclonal antibodies, KIM127 and m24, respectively (26) \u003cstrong\u003e(Fig. 2a)\u003c/strong\u003e.Human primary CD4 T cells SSH1 expression was targeted by small interfering RNAs (siRNAs) against the \u003cem\u003eSSH1\u003c/em\u003e gene (siSSH1) or unspecific siRNA (siCTRL) (\u003cstrong\u003eExtended Data Fig. 1a). \u003c/strong\u003eCells were allowed to spread over anti-CD3 and anti-CD28 antibodies-coated plates and analyzed through flow cytometry. Activated siSSH1 CD4 T cells contained decreased extended-closed \u003cstrong\u003e(Fig. 2b)\u003c/strong\u003e and extended-open \u003cstrong\u003e(Fig. 2c)\u003c/strong\u003e LFA-1, which was also confirmed in \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells (\u003cstrong\u003eExtended Data Fig.1b,c\u003c/strong\u003e). In contrast, VLA-4 activation was unaffected in these cells in similar assays by using HUTS21 antibody, which recognizes an activation-dependent epitope of VLA-4 integrin (27) (\u003cstrong\u003eExtended Data Fig.1d,e\u003c/strong\u003e). The expression of LFA-1 and VLA-4 integrins remained unchanged by loss of SSH1 (\u003cstrong\u003eExtended Data Fig.1f\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003eThe effect of SSH1 absence was explored in depth by studying the spatial distribution of total and active LFA-1 at the IS. In control cells, the majority of detected LFA-1 showed extended-closed conformation and was found at the IS \u003cstrong\u003e(Fig. 2d)\u003c/strong\u003e, in the internal side of the F-actin ring \u003cstrong\u003e(\u003c/strong\u003e3D in\u003cstrong\u003e Fig. 2e)\u003c/strong\u003e, while low extended-close LFA-1 was observed in SSH1 defective cells, with an aberrant localization of LFA-1 at more external areas of the F-actin ring \u003cstrong\u003e(Fig. 2d,e)\u003c/strong\u003e. To confirm the aberrant localization of LFA-1 integrin at the IS regarding the F-actin ring, specific quantifications were performed in control and \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells, finding increased LFA-1 co-localizing with F-actin in \u003cem\u003eSSH1\u003c/em\u003e KO cells (\u003cstrong\u003eExtended Data Fig.1g-j)\u003c/strong\u003e. In addition, in control cells, the more active, extended-open LFA-1 was found at more internal localizations of the F-actin ring, with low LFA-1 molecules stained with the specific antibody, while \u003cem\u003eSSH1\u003c/em\u003e KO cells showed active LFA-1 localized at F-actin regions \u003cstrong\u003e(Fig. 2f,g).\u003c/strong\u003e These data point to decreased adhesion in the absence of SSH1. In this regard, although VLA-4 activation was not affected when studied through flow cytometry (\u003cstrong\u003eExtended Data Fig.1d,e\u003c/strong\u003e), its active form showed an altered distribution in the synapse (\u003cstrong\u003eExtended Data Fig.1k,l\u003c/strong\u003e), suggesting a general role for SSH1 in integrin localization at the IS upon activation, probably through promoting the transmission of the inside-out signaling from the TCR to the cytoskeleton and facilitating the local, contractile activity of Myosin IIA. In support of this, the phosphorylation and activation of Pyk2 pY402 and Vav1 pY174 were decreased in siSSH1 CD4 T cells \u003cstrong\u003e(Fig. 2h,i\u003c/strong\u003e) and \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells, which showed similar defects in Myosin-IIA activation by means of decreased myosin light-chain phosphorylation, as observed for siSSH1 cells (\u003cstrong\u003eExtended Data Fig.1m,n\u003c/strong\u003e). Vav1 and Pyk2 are relevant mediators for inside-out signaling by connecting TCR to actin cytoskeleton remodeling and integrin clustering and by regulating the position of the centrosome at the IS, which may be also regulated by actin dynamics (1,28). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eSSH1 promotes tubulin dynamics after TCR activation \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eActin and microtubules are both critical for integrin activation, which confer stability to the synapse, and force transmission through ADAP and Myosin-IIA (29,30). This can be required to move the centrosome, together with proper control of F-actin at the centrosome area (31), which could be altered by loss of SSH1. In this regard, primary siSSH1 CD4 T cells did not polarize their centrosome to IS \u003cstrong\u003e(Fig. 3a,b).\u003c/strong\u003e We therefore studied whether centrosomal actin in JK-Raji conjugates was correctly regulated in \u003cem\u003eSSH1\u003c/em\u003e KO cells \u003cstrong\u003e(Fig. 3c)\u003c/strong\u003e\u003cstrong\u003e, \u003c/strong\u003ewhich showed a decreased centrosome polarization index \u003cstrong\u003e(Fig. 3d)\u003c/strong\u003e,concomitant with an increased centrosomal F-actin index \u003cstrong\u003e(Fig. 3e)\u003c/strong\u003e. This resulted in the loss of correlation between both indexes \u003cstrong\u003e(Fig. 3f) \u003c/strong\u003ecompared with control JK T cells \u003cstrong\u003e(Fig. 3g)\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003eSSH1-silenced primary CD4 T cells also showed increased F-actin surrounding the centrosome (PCM-1 centered in tubulin in maximal projections) upon activation, which can be observed in 3D together with a defect in the clearance of actin at the central part of the IS \u003cstrong\u003e(Fig. 3h). \u003c/strong\u003esiSSH1 CD4 T cells showed increased F-actin index at the centrosomal area \u003cstrong\u003e(Fig. 3i\u003c/strong\u003e). Indeed, this F-actin seems to arise from the non-polarized Golgi complex, which surrounds the centrosome, in siSSH1 CD4 T cells, connecting Golgi with the IS area \u003cstrong\u003e(Fig. 3h\u003c/strong\u003e,\u003cstrong\u003ej). \u003c/strong\u003eGolgi distance to the IS was consequently increased in IS formed by \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells with APCs (\u003cstrong\u003eExtended Data Fig. 2a,b\u003c/strong\u003e). In SSH1-defective cells, the excess of F-actin at the centrosomal area may interfere with the localization of the centrosome at the IS, preventing its relocation toward the IS. Since the orientation of the centrosome can rescue defective centrosomal polarization in terms of the organization of the microtubule network at the IS (32),westudied upstream signaling for microtubule regulation such as LIMK1 and Aurora kinase A, which is upstream of PKC during T cell activation and required for centrosomal positioning (33). Cells defective for SSH1, either JK or primary CD4 T cellsshowed increased phosphorylation of LIMK1/2 pT508/505 \u003cstrong\u003e(Fig. 3k,l, Extended Data Fig. 2c,d\u003c/strong\u003e), as well as Aurora A phosphorylation at T288 \u003cstrong\u003e(Fig. 3k,l, Extended Data Fig. 2c,d\u003c/strong\u003e). To assess tubulin dynamics at the IS, live-cell total internal reflection fluorescence microscopy (TIRFm) was used to track EB3-GFP decorated plus-tips of microtubules in \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells during IS. Control cells were able to polarize the centrosome near the IS in these assays while \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells showed reduced microtubule dynamics \u003cstrong\u003e(Fig. 3m,n, Supplementary Video 1)\u003c/strong\u003e. This was reflected by the loss of acetylation kinetics of \u0026alpha;-tubulin at lysine 40 (K40-\u0026alpha;-tubulin) upon IS formation (\u003cstrong\u003eFig. 3o)\u003c/strong\u003e, that did not decrease rapidly upon TCR activation as described before (34) and increased in cells forming synapses for extended times (\u003cstrong\u003eExtended Data Fig. 2e,f\u003c/strong\u003e). Therefore, these data point to SSH1 as a regulator of actin dynamics at different locations in activated cells, connecting actin and microtubule dynamics at the IS and helping the intracellular organization of T cells.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMetabolic regulation of T cells is fine-tuned by SSH1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter TCR engagement, mitochondria are transported along microtubules and accumulate around the translocated centrosome and Golgi at the IS, beneath the F-actin ring in a process also regulated by LFA-1 integrin adhesion (6,35-37). There, mitochondria fuel myosin light-chain phosphorylation (38). Therefore, mitochondria, LFA-1 and F-actin were studied through confocal microscopy at the IS established by control and \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells with SEE-loaded Raji cells, finding defective mitochondria positioning at the IS in the absence of SSH1 \u003cstrong\u003e(Fig. 4a,b\u003c/strong\u003e). Likewise, siSSH1 CD4 T cells were unable to polarize mitochondria to the IS \u003cstrong\u003e(Fig. 4c,d, Extended Data Fig. 3a).\u003c/strong\u003e To further assess mitochondria in these cells, we isolated mitochondria from resting or stimulated control and \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells to study their attached regulatory, cytoskeletal and motor components, such as kinesin-1 and cytosolic dynein \u003cstrong\u003e(Fig. 4e,f).\u003c/strong\u003e Mitochondrial-resident SSH1 was found increased in activated control JK T cells together withLIMK, as well as \u0026alpha;-tubulin and \u0026beta;-actin, which were increased in these mitochondria, reflecting their relationship with the cytoskeleton to organize their movement to the IS. In addition, LIMK1/2 seems activated, as it is phosphorylated at pT508/505, and tubulin shows acetylation at Lys 40, indicating stability of microtubules (\u003cstrong\u003eFig. 4e)\u003c/strong\u003e. The lack of SSH1 increased LIMK1 recruitment, as well as acetylation of tubulin and the amount of actin recovered (\u003cstrong\u003eFig. 4e\u003c/strong\u003e), suggesting increased stability of the cytoskeleton around the mitochondria, and augmented docking of the mitochondria in these cells. In this regard, in control cells TCR activation induced the binding of kinesin-1 molecular motor, formed by kinesin heavy chain (KHC) and kinesin-1 light chain (KLC), and moderate recruitment of p74-dynein (cytosolic dynein) \u003cstrong\u003e(Fig. 4f)\u003c/strong\u003e. Kinesin-1, a molecular motor leading movement toward the plus-end of microtubules (36,39), would facilitate mitochondrial transport along microtubules toward the plasma membrane to reach the IS, whereas dynein would transport mitochondria toward the centrosome. In \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells, the kinesin-1 was greatly increased upon activation after T cell stimulation, as well as dynein \u003cstrong\u003e(Fig. 4f)\u003c/strong\u003e. Taken together, these results suggest that SSH1 regulation of actin and tubulin cytoskeleton is required to regulate the interaction of mitochondria with the cytoskeleton and the proper recruitment of the molecular motors involved in their transport. This transport along the cytoskeleton is essential to position mitochondria correctly at the IS (36,39), which is not observed in SSH1-defective cells. Beyond localization, the fate and function of mitochondria depend on cytoskeletal docking (36,39). Therefore, mitochondrial mass was assessed by staining cells with NAO, a mitochondrial probe that binds to cardiolipin in mitochondria, independently of the mitochondrial membrane potential (\u0026Delta;\u0026Psi;m). When compared with control cells, no differences were observed in siSSH1 CD4 T cells \u003cstrong\u003e(Fig. 4g)\u003c/strong\u003e, although \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells exhibited a decrease in mitochondrial mass (\u003cstrong\u003eExtended Data Fig. 3b\u003c/strong\u003e). Mitochondrial ROS was then analyzed, finding that siSSH1 CD4 T cells produced higher amounts of ROS, which were further increased shortly upon T cell activation (5 min) and sustained for several hours, while siCTRL cells showed a more modest increase at 15 min, that dropped in the first hour of activation (\u003cstrong\u003eFig. 4h, Extended Data Fig. 3c)\u003c/strong\u003e. These results indicate that the mitochondrial complexes required for oxidative phosphorylation (OXPHOS) are not correctly responding in SSH1-deficient cells. \u0026Delta;\u0026Psi;m was then analyzed with MitoTracker Deep Red FM normalized to Tom20 in siSSH1 CD4 T cells, which were hyperpolarized, whereas control cells exhibited a drop in \u0026Delta;\u0026Psi;m at 2-15 min after T cell activation, which corresponds to high production of ATP early after TCR activation (39), and then a re-polarization upon 30 min of stimulation (\u003cstrong\u003eFig. 4i, Extended Data Fig. 3d)\u003c/strong\u003e. Increase in \u0026Delta;\u0026Psi;m was corroborated through confocal microscopy in siSSH1 CD4 T cells (\u003cstrong\u003eFig. 4j-l\u003c/strong\u003e) and \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells (\u003cstrong\u003eExtended Data Fig. 3e,f\u003c/strong\u003e) by measuring MitoTracker Orange/Tom20 ratios\u003cstrong\u003e.\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eTo further address mitochondria activity, control and siSSH1 CD4 T cells were subjected to a mitostress test using glucose as external energy source and measuring the oxygen consumption rate (OCR) as indicator of mitochondrial respiration \u003cstrong\u003e(Extended Data Fig. 3g)\u003c/strong\u003e. siSSH1 CD4 T cells showed unchanged basal respiration, with decreased ATP production after TCR stimulation compared with siCTRL CD4 T cells (\u003cstrong\u003eFig. 4m)\u003c/strong\u003e, corroborating our data on mitochondrial hyperpolarization. In addition, inaccordance with the defects observed in the recruitment of the required molecular motors to help reorganize the IS in activated T cells, siSSH1 CD4 T cells did not respond to TCR stimulation by increasing their maximal respiration and spare respiratory capacity as did siCTRL cells (\u003cstrong\u003eFig. 4m\u003c/strong\u003e). In the case of \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells, an exacerbated phenotype was observed, with decreased basal respiration, ATP production and maximal respiration at resting conditions and in response to TCR activation (\u003cstrong\u003eExtended Data Fig. 3h,i\u003c/strong\u003e), probably due to supplementary problems due to long-term knock-down of SSH1, such as decreased mitochondrial mass (\u003cstrong\u003eExtended Data Fig. 3b\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003esiCTRL and siSSH1 CD4 T cells were also challenged to ascertain whether they were able to oxidize glucose through glycolysis, which is known to increase during T cell activation (14). Extracellular acidification rate (ECAR) was used as an indicator of glycolysis by using the Seahorse analyzer \u003cstrong\u003e(Extended Data Fig. 3j)\u003c/strong\u003e. siSSH1 CD4 T cells showed reduced capacity to increase glycolysis and glycolytic capacity upon stimulation with anti-CD3 and anti-CD28 antibodies, with no significant changes without TCR stimulation (\u003cstrong\u003eFig. 4n)\u003c/strong\u003e. \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells exhibited reduced glycolysis and glycolytic capacity both at rest and after TCR stimulation (\u003cstrong\u003eExtended Data Fig.3k,l\u003c/strong\u003e). A reduction in the Akt/mTOR/S6 signaling pathway was observed in siSSH1 CD4 T cells, although significant signaling levels were still present with phosphorylation of Akt, mTOR and S6 (\u003cstrong\u003eFig. 4o,p\u003c/strong\u003e). This decrease was also observed in \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells (\u003cstrong\u003eExtended Data Fig. 3m,n\u003c/strong\u003e) after T cell activation, pointing to reduced response to TCR activation and co-stimulation.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eSSH1 connects organelle localization and metabolic response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough Akt/mTOR signaling can be detected in CD4 T cells expressing low levels of SSH1, their metabolic response seems highly affected. An emerging hypothesis indicates that inter-organelle contacts collaborate in organizing T cell metabolic responses (9). To gain insight into this possibility, the distribution of mitochondria and peroxisomes regarding the tubulin cytoskeleton during IS formation was assessed. Whereas siCTRL CD4 T cells showed re-localization of the centrosome, mitochondria and peroxisomes to the IS, siSSH1 CD4 T cells exhibited defective localization of peroxisomes at the IS (\u003cstrong\u003eFig. 5a,b)\u003c/strong\u003e. \u003cem\u003eSSH1\u003c/em\u003e KO JK CD4 T cells paralleled the redistribution found in siSSH1 CD4 T cells during IS (\u003cstrong\u003eFig. 5c, Extended Data Fig. 4a)\u003c/strong\u003e. siSSH1 CD4 T cells exhibited a phenotype similar to unstimulated CD4 T cells conjugated to control beads, with their mitochondria and peroxisomes distributed throughout the cell (\u003cstrong\u003eExtended Data Fig. 4b\u003c/strong\u003e). The number of peroxisomes was increased in siSSH1 CD4 T cells (\u003cstrong\u003eFig. 5d,e\u003c/strong\u003e), as well as in \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells (\u003cstrong\u003eExtended Data Fig. 4c-e\u003c/strong\u003e). In addition, peroxisomes proximity to mitochondria increased in siSSH1 CD4 T cells, showing augmented co-localization (\u003cstrong\u003eFig. 5d,f\u003c/strong\u003e), with similar results in \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells\u003cstrong\u003e (Extended Data Fig. 4c,f\u003c/strong\u003e). Further investigation by flow cytometry also showed increased peroxisomes in \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells (\u003cstrong\u003eFig. 5g, Extended Data Fig. 4g\u003c/strong\u003e). Since peroxisomes regulate \u0026beta;-oxidation of long-chain fatty acids until they can be oxidized by mitochondria (40), the increased interaction between mitochondria and peroxisomes and the increase in peroxisomes point to a shift to fatty acid catabolism in SSH1 defective cells.\u003c/p\u003e\n\u003cp\u003eTo probe this hypothesis, neutral lipids were studied by Bodipy 493/503 probe through flow cytometry in \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells. These cells exhibited higher MFI, suggesting increased neutral lipids in cells (\u003cstrong\u003eFig. 5h\u003c/strong\u003e), the main component of lipid droplets (LD), composed of a core of neutral lipids surrounded by a single layer of phospholipids (41). These results, together with the increased peroxisomes (\u003cstrong\u003eFig. 5g\u003c/strong\u003e) and mitochondrial ROS production (\u003cstrong\u003eFig. 4h\u003c/strong\u003e) in SSH1-deficient CD4 T cells, prompted the study of lipid peroxidation in these cells. We used a specific probe that changes its emission wavelength from \u0026sim;590 nm to \u0026sim;510 nm when neutral lipids are peroxidized. These assays revealed that lipid peroxidation increased in \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells (\u003cstrong\u003eFig. 5i, Extended Data Fig.4h\u003c/strong\u003e). LD localization, number and proximity to mitochondria and peroxisomes, known to establish inter-organelle contacts (42), were studied in CD4 T cells and in JK CD4 T cells through confocal microscopy. SSH1 depletion increased the number of LDs per cell (\u003cstrong\u003eFig. 5j,k, Extended Data Fig.4i,-k), \u003c/strong\u003eas well asthe number of LD contacts with peroxisomes and mitochondria (\u003cstrong\u003eFig. 5j,l,m, Extended Data Fig.4i,l,m)\u003c/strong\u003e, consistent with defective polarization of LDs to the IS formed by \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells and SEE-loaded Raji B cells\u003cstrong\u003e (Extended Data Fig. 4j,n\u003c/strong\u003e). This supports a differential use of LDs in SSH1-silenced cells. Therefore, endogenous fatty acid oxidation (FAO) was determined in siCTRL and siSSH1 CD4 T cells. CD4 T cell activation induced FAO, and defective SSH1 expression led to a 50% increase in FAO in resting conditions, which is further reinforced in response to TCR and co-stimulation (\u003cstrong\u003eFig. 5n, Extended Data Fig. 4o-p\u003c/strong\u003e). This response was enhanced when palmitate was provided as external source in similar assays, showing a 100% increase in FAO in resting conditions, further augmented by activation in siSSH1 CD4 T cells (\u003cstrong\u003eFig. 5o, Extended Data Fig. 4p\u003c/strong\u003e). \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells showed a similar increase in FAO, under basal conditions and following TCR stimulation, both during oxidation of endogenous fatty acids and in the presence of exogenous palmitate (\u003cstrong\u003eExtended Data Fig. 4q-t\u003c/strong\u003e). In addition, SSH1-silenced cells showed sustained OCR when etomoxir was injected, while OCR fell dramatically in control cells \u003cstrong\u003e(Extended Data Fig. 4u\u003c/strong\u003e). This is consistent with carnitine palmitoyltransferase I (CPT1)-independent FAO pathways, such as peroxisomal \u0026beta;-oxidation, and aligns with our data of lipid peroxidation (\u003cstrong\u003eFig. 5i\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003eThis suggests an augmented peroxisomal FAO capacity due to the observed increased LD-peroxisome-mitochondria contacts and peroxisome number. These data demonstrate that SSH1 controls organelle distribution and localization at the IS, helping mitochondrial respiration and glycolysis shift, and that SSH1 absence enhances lipid metabolism through LD increase and regulation of their contacts with peroxisome and mitochondria (\u003cstrong\u003eFig. 6\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study identifies SSH1 phosphatase as a central coordinator of actin and tubulin cytoskeleton crosstalk and LFA-1 integrin activation through the establishment of a protein hub facilitating proper organelle positioning during T cell activation. Hence, our data show that SSH1 bridges key integrin regulators Talin-1, Kindlin-3 and ADAP (18,\u0026nbsp;20) at the IS and facilitates the transition of LFA-1 into its high-affinity state (\u003cstrong\u003eFig. 6).\u003c/strong\u003e Talin-1, Kindlin-3 and ADAP, stabilize high-affinity LFA-1 clusters at the IS (19) allowing transduction of TCR signals to integrin activation, helping inside-out signaling (18). In this context, the described SSH1-driven actin severing and turnover (11) may promote Talin-1 binding and tensile actin flow that separate the LFA-1 integrin \u0026alpha;/\u0026beta; tails, which is the final inside-out step of LFA-1 integrin activation (43, 44). The described complex in this study extends the role of SSH1 in the control of T cell actin remodeling, since SSH1 allows Talin-1 and Kindlin-3 to interact with ADAP and Myosin IIA, ultimately promoting complete LFA-1 integrin activation and localization, mainly at the more internal area of the F-actin ring formed in the lamella of T cells organizing IS. There, SSH1-deficient cells showed reduced LFA-1 in extended-closed, and especially in extended-open conformations. These findings underscore SSH1 as a key molecule for building high-avidity LFA-1 adhesions at the synapse. In this regard, actin-driven forces are known to stabilize high-affinity LFA-1, and tensile stress orients the integrin on the membrane and locks its headpiece open (45). Our results show that SSH1 and Myosin IIA form part of a regulated complex that is fostered by T cell activation via TCR, bringing together Talin-1, Kindlin-3 and ADAP with Myosin IIA activity. These data suggest that SSH1 acts as a scaffold protein, maintaining the complex hub for integrin activation and the inside-out integrin TCR-derived signaling required for this activation, as observed in the case of phosphorylation and activation of Pyk2 (pY402) and Vav1 (pY174), and Myosin IIA through the phosphorylation of myosin regulatory light chain (MLC2) pT18/pS19, required for \u0026nbsp;tension generation and mechanotransduction (22-24). This action seems specific to LFA-1, since VLA-4 integrin activation is not affected in SSH1-deficient T cells. This could be explained because of the differential location of these integrins at the IS. VLA-4 integrin acts distally, while LFA-1 acts at the lamella. In this regard, beta-2 integrins, such as LFA-1, in contrast to beta-1 integrins, such as VLA-4, are required to complete the centripetal transport of SLP-76 microclusters at the IS in a process mediated by contractile Myosin IIA (30). In this regard, SLP76 phosphorylation at pY145 was previously shown to be hampered in SSH1-depleted T cells (10).\u003c/p\u003e\n\u003cp\u003eBeyond actin, which can be regulated by SSH1‐induced cofilin activation (11,13), thereby preventing excessive F-actin rigidity and accumulation at the IS (10), SSH1 activity also impacts microtubule dynamics at the synapse. Interestingly, loss of SSH1 led to increased F-actin at the centrosome area and major tubulin acetylation, impaired centrosome and Golgi polarization to the IS, and lowered microtubule growth when the centrosome reached the IS (\u003cstrong\u003eFig. 6\u003c/strong\u003e). \u0026nbsp;Recently, F-actin at the centrosome area was shown to decrease upon TCR activation, and this is relevant for IS formation, not only in T cells, but also in B cells (46). This is due to the requirement for F-actin clearance at the centrosome area to allow centrosome polarization and directed secretion at the IS (47,48), in a process regulated by PKA (49). This observation is consistent with the increased F-actin accumulation at the IS in SSH1-deficient T cells (10). Golgi complex is also found far from the IS in SSH1-deficient cells, since the reorientation of the Golgi and associated vesicles depends on the correct localization of the centrosome at the IS (50). This is also relevant to sustain long-term TCR activation, thanks to the polarized delivery of signaling molecules at the IS, such as LAT, a relevant scaffold (51). In this context, proper orientation of the centrioles and the centrosome is crucial for the organization of the microtubular network at the IS (32), and centrosome localization at the IS is also supported by tubulin dynamics. This is supported by early HDAC6 activity, promoting deacetylation of pre-existing MTs a few minutes before T cell activation, which coincides with centrosome translocation toward the IS (34), kinesin activity (52) and later increase in MT stabilization after polymerization, since acetylated tubulin at lysine 40 is a marker of microtubule stability (53). Concerning this, SSH1-deficient T cells showed a sustained increase in K40-acetylated-\u0026alpha;-tubulin during T cell activation, with a concomitant defect in microtubule growth and dynamics observed by live-cell TIRFm. In addition, Aurora A kinase, which is phosphorylated at T288 and activated early upon TCR activation and regulates Lck activity, helps centrosome translocation toward the IS and MT growth to enable TCR early signaling, intracellular trafficking at the IS (31), as does its downstream effector, Polo-like kinase 1 (Plk1), regulating target cell killing (54). Interestingly, increased Aurora A phosphorylation at T288 augmented T cell signaling in transgenic mice (33), but not in SSH1-deficient CD4 T cells, where exacerbated phosphorylation of Aurora A did not rescue centrosome polarization. This also happens with increased phosphorylation of LIMK1/2 pT508/505 (10) and this study. Indeed, LIMK is involved in actin-microtubule crosstalk through the regulation of Aurora A by phosphorylation (55). These results indicate that SSH1-deficient cells show defects that cannot be rescued by increased activity of kinases, that probably rely on differential positioning of proteins in complexes in a SSH1-dependent manner and warrant further research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCentrosome translocation toward the IS establishes the site of Golgi localization, and both allow the trafficking of vesicles and mitochondria toward the IS (36,56). In T lymphocytes, mitochondria preferentially localize near the IS during T cell activation, in a process regulated by Drp1 through actomyosin-dependent centripetal flux control (36). At the IS, mitochondria regulate calcium signaling (57,58) and orchestrate IS stability through ATP production via oxidative phosphorylation (39), two important factors needed for T cell activation (8). The transport and localization of mitochondria toward the IS not only depend on integrin adhesion (6), but also on tubulin cytoskeleton in collaboration with kinesin and dynein motor proteins (34,58). In addition, CXCL12 chemokine induced inside-out activation of LFA-1 can even pre-polarize the centrosome and mitochondria toward a prospective synapse independently of TCR signals, priming the T cell for robust Ca\u003csup\u003e2+\u003c/sup\u003e influx and NFAT signaling upon antigen recognition (6). Concerning this, SSH1 overexpression in JK T cells leads to permanent activation of cofilin and suppression of CXCL12-mediated directed migration (59). Together, these observations align with our data showing that SSH1-deficient T cells exhibit an impaired activation and localization of LFA-1 integrin, but also an impaired centrosome polarization and Golgi orientation, which could explain the hampered polarization of mitochondria at the IS. Moreover, in migrating T cells, mitochondria redistribution, as well as the centrosome, depends on dynein/dynactin complex in collaboration with Miro-1, an adaptor molecule that couples mitochondria to microtubules (60,61). This is consistent with the increased levels of kinesin and actin observed in isolated mitochondria from TCR-stimulated \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells, which may cause an imbalanced or inhibited transport of these organelles after T cell activation\u0026nbsp;due to altered stoichiometry of motor/regulatory protein complexes. It has been previously described that mitochondrial localization is critical for its function (8,58,62). Although homeostatic ROS production is determinant for TCR derived signaling and NFAT translocation to the nucleus promoting IL-2 production (63), mitochondrial ROS overproduction has been associated with T cell senescence (64) and systemic lupus erythematosus (65). A similar observation has been reported for mitochondrial membrane potential, since mitochondria hyperpolarization, together with an increased mitochondrial ROS production and a defective ATP production, has been reported in T cells from human type 1 diabetes and systemic lupus erythematosus patients (65,66). This evidence supports the altered localization of mitochondria in SSH1-deficient cells as a mechanism to explain the defect in mitochondrial function observed in these cells, with increased ROS production and \u0026Delta;\u0026Psi;m, resulting in affected OXPHOS.\u0026nbsp;This effect may be due to defects in the Akt/mTOR/S6 signaling pathway, which is involved in glucose-derived metabolism and ribosomal synthesis in T cells (67), in SSH1-silenced T cells, although signaling was still present. Thereby, other mechanisms can be acting in this scenario. In this regard, other metabolic organelles are mobilized together with mitochondria, such as peroxisomes and LDs that polarized to the IS in activated T cells, reflecting a broader organelle orchestration that accompanies IS formation (68). The polarization of mitochondria and peroxisomes to the IS has been proposed as a mechanistic link between the metabolic shifts and the signaling network in T cells, with important implications in cancer immunotherapy (9). Peroxisomes cooperate with mitochondria and LDs to channel FA into oxidative pathways and their relocation in activated T cells may facilitate efficient energy production and redox homeostasis required for proper sustained signaling (69). Concerning this, SSH1-deficient cells fail to polarize peroxisomes to the IS, while their number and interaction with mitochondria are increased together with ROS production in SSH1-deficient CD4 T cells. We propose that SSH1, by linking actin turnover at different locations in cells, and by bridging Talin-1/ADAP complexes and Myosin IIA, creates functional local actin scaffolds necessary to regulate actin dynamics and dynamic microtubule network at different locations, such as F-actin at the IS, at the centrosome area and even at the mitochondria. The resulting actin\u0026ndash;microtubule crosstalk ensures the function of molecular motors, such as Myosin IIA and kinesin-1 in the reorientation of organelles such as mitochondria, peroxisomes and LDs and the establishment of relevant contacts to regulate their activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFatty acid metabolism is concurrently reprogrammed in T cells, with increased lipid synthesis and enhanced FAO, supporting T cell differentiation and long-term survival (70, 71). Although lipid metabolism and FAO play an important role in the differentiation and maintenance of memory T cells (70, 71), an excessive increase in both lipid accumulation and utilization could alter short-term T cell activation, promoting an exhausted T cell phenotype, characterized by lipotoxicity and lipid peroxidation (72). In this regard, our findings reveal that SSH1 control of the synapse extends to T cell lipid metabolism. SSH1-deficient T cells display excessive lipid droplets accumulation, increased lipid peroxidation, enhanced LD-peroxisome\u0026ndash;mitochondria contacts, and elevated FAO, suggesting a shift toward lipid oxidative metabolism. Furthermore, SSH1-silenced T cells showed sustained OCR after injection of etomoxir, an inhibitor of CPT1 transport, which blocks the entry of FA into mitochondria, thereby inhibiting FAO. This suggests that SSH1-silenced cells have a compensatory mechanism, in which junctions between LDs and peroxisomes allow the latter to directly access fatty acids stored in LDs for \u0026beta;-oxidation in peroxisomes. Recent findings reveal that LD trafficking to peroxisomes is essential for maintaining mitochondrial metabolism (73,74), and that the impairment in the crosstalk between mitochondria and endoplasmic reticulum (ER) impedes the use of glucose-derived pyruvate as mitochondrial fuel, causing a shift to FA to sustain energy production (75). In addition, the interaction between LDs, ER and mitochondria helps the recruitment of peroxisomes to the organelle-metabolic hub, supporting FA efflux from LD and enhancing lipid metabolism (76). \u0026nbsp;Regarding this, the increased contacts between these three organelles in SSH1-deficient cells likely create an integrated network of lipid catabolism: fatty acids can be mobilized from LDs and transferred directly to peroxisomes and mitochondria. Overall, increased contacts between LDs, peroxisomes, and mitochondria in SSH1-deficient T cells are expected to enhance FAO, explaining why these cells maintain OCR better even when mitochondrial FA import is pharmacologically blocked with etomoxir. We hypothesize that robust synapse formation via SSH1 couples to mitochondrial and glycolytic glucose fueling of effector functions, whereas loss of SSH1 forces cells to rely more on FAO to meet energy demands. This concept fits emerging paradigms: activated T cells reposition mitochondria to the synapse where they buffer Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003eand supply ATP for actomyosin contractility, while peroxisomes and lipid stores cooperate with mitochondria to sustain redox homeostasis.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In summary, our data position SSH1 as a key regulator of T cell activation, enabling TCR-driven actin remodeling that primes LFA-1 for high-affinity adhesion, while simultaneously fostering dynamic microtubules and organelle re-localization to meet the biosynthetic needs of activation. This integrated mechanism, coupling cytoskeletal reorganization to metabolic shift, ensures that signal transduction, adhesion and energy supply progress in concert (\u003cstrong\u003eFig. 6\u003c/strong\u003e). By focusing on the role of SSH1, we provide novel insight on actin dynamics feed into integrin activation and synapse maturation, which may serve to modulate T cell function.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eCell lines and human CD4 T cell isolation from peripheral blood\u003c/p\u003e\n\u003cp\u003eThe human Jurkat E6-1 CD4 T cell line (V\u0026alpha;l.2 V\u0026beta;8\u003csup\u003e+\u003c/sup\u003e TCR) and the lymphoblastoid B cell line Raji (Burkitt lymphoma; obtained from the DSMZ Organization; ACC-319) were cultured in RPMI 1640 + GlutaMAX\u0026ndash;I + 25\u0026thinsp;mM HEPES (Gibco\u0026ndash;Invitrogen) and supplemented with 10% fetal bovine serum (FBS) (Hyclone, Thermofisher). All lymphoid cell lines were routinely tested for specific expression of CD (clusters of differentiation) with specific antibodies by flow cytometry and for mycoplasma infection by PCR. \u003cem\u003eSSH1 \u003c/em\u003eKO cells were generated using CRISPR\u0026ndash;Cas9 method as described (10).\u003c/p\u003e\n\u003cp\u003eHuman peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors by separation on a Biocoll gradient (Biochrom) according to standard procedures. CD4 T cells were purified from PBMCs using an EasySep negative isolation kit for human CD4 T cells (17952; Stem Cell Technologies). CD4 T cells were nucleofected with control or SSH1-specific siRNAs (2.5 \u0026micro;M) in Opti-MEM I (Gibco-Invitrogen)\u003csup\u003e \u003c/sup\u003eand used for activation assays 48 h post-transfection. These studies were performed adhered to the principles of the Declaration of Helsinki and were approved by the local ethics committee for basic research at the \u0026lsquo;\u003cem\u003eHospital La Princesa\u003c/em\u003e\u0026rsquo;. Informed consent was obtained by Centro Transfusiones Comunidad Autonoma de Madrid (CAM).\u003c/p\u003e\n\n\u003cp\u003eAntibodies, reagents and probes\u003c/p\u003e\n\u003cp\u003eThe commercial primary antibodies used in this study were anti-phospho-Aurora A T288 (ab83968; 1:500 for WB), anti-Aurora A (a13824; clone 35C1; 1:500 for WB), anti-GLG1 Golgi complex (ab103439; 1:100 for IF) and anti-GFP (ab13970; 1:200 for IF) from Abcam; anti-ADAP/SLAP-130/Fyb (07-546; 1:500 for WB, 1:50 for IP), anti-\u0026beta;-actin (AM4302; clone AC-15; 1:2,000 for WB), anti-\u0026alpha;-tubulin (T6199; clone DM1A; 1:2,000 for WB), anti-(Lys40)-Acetyl-\u0026alpha;-tubulin (32-2700; clone 12B4; 1; 1:2,000 for WB, 1:1,000 for IF) and fluorescein isothiocyanate (FITC)-conjugated anti-\u0026alpha;-tubulin (F2168; clone DM1A; 1:100 for IF) from Sigma Aldrich; anti-Kinesin Heavy Chain (MAB1613;clone H1; 1:500 WB), anti-Kinesin Light Chain (MAB1617; clone L1; 1:500 for WB) and anti-Dynein Intermediate Chains (MAB1618; clone 74.1; 1:500 for WB) were from Merck Millipore; anti-phospho-LIMK1/2 Y507/T508 (07-850; 1:500 for WB), anti-phospho-Pyk2 Y402 (44-618G; 1:1,000 WB), anti-VDAC (PA1-954A; 1:500 for WB), anti-Pex14 (PA5-78103; 1:100 for IF, 1:100 for FACS), anti-Myosin-IIA (A304-490A; 1:2,000 for WB, 1:200 for IP) and anti-Talin-1 (14168-1-AP; 1:1,000 for WB, 1:100 for IP, 1:100 for IF) were from ThermoFisher-Invitrogen; anti-CD4-PE (317410; clone OKT4; 1:200 for FACS), anti-CD4-APC (317416; clone OKT4; 1:200 for FACS) and anti-CD3\u0026epsilon; (clone HIT3a) for stimulatory surfaces were from BioLegend. anti-p150\u003cem\u003e\u003csup\u003eglued\u003c/sup\u003e \u003c/em\u003e(610474; 1:500 for WB), anti-CD28 (555726; clone CD28.2; 6.67 \u0026mu;g/mL) for stimulatory surfaces and anti-PKC\u0026theta; (610090; 1:500 for WB) were from BD Pharmingen; anti-Perilipin 2 (GP46; 1:50 for IF) was from Progen; anti-Tom20 (11802-1-AP; 1:2000 for WB, 1:500 for IF) was from Proteintech; anti-phospho-mTOR S2448 (2971S;1:1,000 for WB), anti-mTOR (2972S;1:1,000 for WB), anti-phospho-S6 S235/S236 (2211S;1:1,000 for WB), anti-S6 ribosomal subunit (2217S;1:1,000 for WB), anti-phospho-Akt S473 (9271S;1:1,000 for WB), anti-Akt (9272S;1:1,000 for WB), anti-phospho-MLC2 T18/S19 (3674S;1:1,000 for WB), anti-MLC2 (3672S;1:1,000 for WB), anti-phospho-PKC\u0026theta; T538 (9377S;1:1,000 for WB), anti-Kindlin-3 (10459S; 1:1,000 for WB, 1:100 for IP, 1:100 for IF), anti-PCM-1 (5259S; 1:100 for IF) and anti-SSH1 (13578S;1:1,000 for WB, 1:100 for IP) were from Cell Signaling Technology; anti-LIMK1 (sc-515585; 1:200 for WB) and anti-SSH1 (sc-517226; 1:20 for IF) were from Santa Cruz Biotechnology. Ghost Dye Red 780 (13-0865; 1:500 for FACS) and Ghost Dye Violet 510 (13-0870; 1:500 for FACS) were from Tonbo Biosciences.\u003c/p\u003e\n\u003cp\u003eThe following mAbs: anti-m24 and anti-KIM127 mAbs which recognize the activation-dependent epitopes of LFA-1 integrin and anti-HUTS-21, which recognizes the activation-dependent epitope of VLA-4 integrin, have been described previously (26,27); anti-CD11a (clone TP1/40) and anti-CD49d (clone HP2/1) to detect total LFA-1 (\u0026alpha;L) and VLA-4 (\u0026alpha;4) integrins, respectively, were produced in our laboratory (77).\u003c/p\u003e\n\u003cp\u003eThe anti\u0026ndash;phospho-Vav Y174 mAb was a kind gift from Dr X. Bustelo (Centro de Investigaci\u0026oacute;n del C\u0026aacute;ncer). Cell tracker CMAC (7-amino-4-chloromethylcoumarin; 10 \u0026mu;M ,C2110) was from Molecular Probes, Invitrogen; SEE (0.5\u0026thinsp;\u0026mu;g/mL, PE404) was from Toxin Technologies; Prolong gold antifade mounting medium (P-36934), prolong gold antifade mounting medium with DAPI (P-36931), Dynabeads M-280 sheep anti-rabbit IgG (11203D) and Dynabeads M-280 sheep anti-mouse IgG (11202D) were from ThermoFisher Scientific; fibronectin and poly-L-Lysine were from Sigma Aldrich; recombinant ICAM-1 was from H\u0026ouml;lzel Diagnostika Handels GmbH; anti-CD3/anti-CD28 antibodies (ImmunoCult human T cell activator; 10991) and human recombinant IL-7 (78053) were purchased from Stem Cell Technologies.\u003c/p\u003e\n\u003cp\u003eReagents and probes were as follows: MitoTracker Orange CMTMRos (M7510; 500 nM for IF), Nonyl-acridine orange (NAO; A1372; 25 nM for FACS) for mitochondrial mass determination, MitoTracker Deep Red FM (M22426, 500 nM for FACS) for mitochondrial membrane potential quantification, MitoSOX Green (M36006; 1 \u0026micro;M for FACS) for ROS determination, BODIPY 581/591 C11 (D3861; 0.8 \u0026micro;M for FACS) for lipid peroxidation detection and BODIPY 493/503 (D3922; 0.8 \u0026micro;M for FACS) for neutral lipids quantification were from Life Technologies-Invitrogen. \u003c/p\u003e\n\u003cp\u003eThe following secondary reagents were used: phalloidin conjugated to Alexa Fluor 647 (A-22287; 1:40 for IF), phalloidin conjugated to Alexa Fluor 488 (A-12379; 1:40 for IF), goat anti-rabbit and goat anti-mouse highly cross-adsorbed secondary antibodies conjugated to Alexa Fluor 488 (A-11034 and A-11029, respectively; 1:500 for IF), 568 (A11036 and A-11031, respectively; 1:500 for IF) or 647 (A-21443 and A-21236, respectively; 1:500 for IF), donkey anti-goat highly cross-adsorbed secondary antibody conjugated to Alexa Fluor 647 (A-21447; 1:500 for IF), donkey anti-rabbit secondary antibody conjugated to Alexa Fluor 555 (A-31572; 1:500 for IF), goat anti-chicken antibody conjugated to Alexa Fluor 488 (A-11039; 1:500 for IF) and goat anti-mouse IgG highly cross-adsorbed secondary antibody conjugated to PE (M30004-1; 1:500 for FACS) were purchased from ThermoFisher Scientific; horseradish peroxidase(HRP)-conjugated secondary antibodies for WB (anti-rabbit 31460, anti-mouse 31430 or anti-goat IgG+IgM 31460; all 1:5,000) were purchased from ThermoFisher Scientific. Rabbit TrueBlot ULTRA: anti-rabbit IgG HRP (18-8816-31; 1:1,000) and mouse TrueBlot ULTRA: anti-mouse IgG HRP (18-8817-30; 1:1,000) were from Rockland. Fluorescence-labelled secondary antibodies IRDye 680 goat anti-rabbit and IRDye 800 goat anti-mouse (926-68071 and 926-32350, respectively; 1:5,000 for WB) were from LI-COR Bioscience.\u003c/p\u003e\n\n\u003cp\u003ePlasmids and siRNA Transfections\u003c/p\u003e\n\u003cp\u003eDouble-stranded control (UUCUCCGAACGUGUGCACG and CGUGCACACGUUCGGAGAA) and SSH1-specific (CGGAGAACCUAAACAACAA and UUGUUGUUUAGGUUCUCCG) siRNAs were purchased from Eurogentec. EB3-GFP plasmid was a kind gift from A. Akhmanova (Utrecht University, Netherlands).\u003c/p\u003e\n\u003cp\u003eFor Jurkat T cell transfection, cells were centrifuged at 1200\u0026thinsp;rpm for 5\u0026thinsp;min, washed with Hank\u0026rsquo;s balanced salt solution (HBSS, Lonza) and resuspended in Opti-MEM I (Gibco\u0026ndash;Invitrogen) (1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells in 400\u0026thinsp;\u0026mu;L). 10\u0026thinsp;\u0026micro;g of plasmids or 2.5 \u0026mu;M siRNAs were added to the cell suspension, which was electroporated in a Gene-Pulse III system (Bio-Rad) set at 240\u0026thinsp;V, 975\u0026thinsp;m\u0026Omega;. After electroporation, cells were resuspended in 5\u0026thinsp;mL RPMI 1640 + GlutaMAX\u0026ndash;I + 25\u0026thinsp;mM HEPES medium + 5% FBS, plated in 25 cm\u003csup\u003e2\u003c/sup\u003e flasks and supplemented with 10% FBS after 3 h. Experiments were performed 24\u0026thinsp;h post-transfection.\u003c/p\u003e\n\u003cp\u003ePrimary CD4 T cells (5 x 10\u003csup\u003e6\u003c/sup\u003e cells in 100 \u0026mu;L) were nucleofected with control or SSH1-specific siRNAs (2.5 \u0026mu;M) in pre-warmed Opti-MEM I (Gibco-Invitrogen) using the U-04 programme of Nucleofector I (Amaxa) after a heat-shock step with cold HBSS. Cells were cultured for 48 h in RPMI 1640 supplemented with 10% FBS and IL-7 (10 ng/mL). Dead cells were discarded using Biocoll Separating Solution 24 h post-transfection. Experiments were performed 48 h post-transfection; specific silencing was verified by Western blot (Extended Data Fig. 1a).\u003c/p\u003e\n\n\u003cp\u003eT cell activation and lysis for immunoprecipitation (IP) and immunoblotting\u003c/p\u003e\n\u003cp\u003eFor human primary CD4 T cell or Jurkat activation with antibodies, 1 x 10\u003csup\u003e6 \u003c/sup\u003ecells per condition in 100 \u0026mu;L of RPMI were stimulated with 20 \u0026mu;L \u0026alpha;CD3\u0026alpha;CD28 antibodies (ImmunoCult Human CD3/CD28 T-Cell Activator; Stem Cell Technologies) for the indicated times. For JK-Raji conjugate formation, 1 x 10\u003csup\u003e5 \u003c/sup\u003eRaji B cells were pulsed with 0.5\u0026thinsp;\u0026mu;g/mL SEE (1 h, 37\u0026ordm;C, complete medium), washed and mixed with 1x10\u003csup\u003e6 \u003c/sup\u003eJurkat E6-1 T cells (1:10) for the indicated times. Cells were centrifuged at 800 rpm at 37\u0026deg;C to promote conjugation. 1 x 10\u003csup\u003e6\u003c/sup\u003e JK T cells were lysed in 50 \u0026mu;L of 5\u0026thinsp;mM Tris-HCl pH 7.5 containing 1% NP40, 0.2% Triton X-100, 150\u0026thinsp;mM NaCl, 2\u0026thinsp;mM EDTA, 1.5\u0026thinsp;mM MgCl\u003csub\u003e2\u003c/sub\u003e with phosphatase and protease inhibitors (Phosphostop and Complete tablets from Roche, respectively) for 30 min on ice followed by a preclearance step by centrifugation at 14,000\u0026thinsp;rpm (4\u0026deg;C, 10\u0026thinsp;min) to remove debris and nuclei. Samples were processed for SDS-PAGE, transferred to nitrocellulose membranes, blocked with TBS containing 0.2% Tween and 5% BSA and incubated with appropriate primary (o/n, 4\u0026deg;C) and peroxidase-labelled secondary antibodies (1 h, RT). Chemiluminescence was detected using the Amersham 880 detection system (GE Healthcare). For fluorescent Western blot, IRDye 680 goat anti-rabbit and IRDye 800 goat anti-mouse secondary antibodies (Li-Cor Biosciences) were also incubated for 1 h at R/T and detected using the Odyssey Infrared Imager (LI-COR Bioscience).\u003c/p\u003e\n\u003cp\u003eFor immunoprecipitation, 1 x 10\u003csup\u003e7\u003c/sup\u003e JK cells per condition were stimulated or not with 50 \u0026mu;L \u0026alpha;CD3\u0026alpha;CD28 antibodies (ImmunoCult Human CD3/CD28 T-Cell Activator; Stem Cell Technologies) in 1 mL of incomplete RPMI for 10 min, centrifuged and lysed in PHEM buffer (60 mM PIPES, 25 mM HEPES, 5 mM EGTA, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e) containing 0.33% Brij 96v supplemented with protease and phosphatase inhibitors for 30 min at RT. Anti‐SSH1 rabbit antibody (10 \u0026micro;g), anti‐Talin-1 rabbit antibody (10 \u0026micro;g), anti-ADAP mouse antibody (10 \u0026micro;g), anti-Myosin IIA rabbit antibody (10 \u0026micro;g) and anti‐Kindlin-3 rabbit antibody (10 \u0026micro;g) were used for immunoprecipitation during 2 h at RT. The complete procedure, including washes and centrifugation, was performed at RT. Preclearing and antibody recovery were performed using 100 \u0026mu;L of dynabeads M-280 sheep anti-rabbit IgG or dynabeads M-280 sheep anti-mouse IgG per IP. Rabbit or mouse serum were used as negative controls (IgG control). Immunoprecipitates and inputs were boiled for 10 min at 85\u0026ordm;C in Laemmli sample buffer 1x containing 5% \u0026beta;-mercaptoethanol and processed for SDS-PAGE. Blots were revealed using True Blot secondary antibodies (1:1,000; Rockland) for detection of primary antibodies and chemiluminescence was detected using the Amersham 880 detection system (GE Healthcare). For the quantification and statistical analysis, immunoprecipitates were normalized by dividing the co-IP prey band intensity by the bait band intensity in the same lane and correcting for the bait which was pulled down. A fold-change was calculated setting unstimulated control value to 1 and dividing all other prey/bait ratios by the control`s ratio (78).\u003c/p\u003e\n\n\u003cp\u003eDensitometric analysis and quantification of Western blots\u003c/p\u003e\n\u003cp\u003eBands from Western blots were quantified (arbitrary units per pixel) using either the supplied Image Gauge (Fujifilm Inc) or Image Studio Lite (v5.2, LI-COR Biosciences) software. Background was subtracted and the resulting values were normalized to unstimulated control samples. The data obtained were statistically analyzed and plotted using PRISM8 (GraphPad software). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCell conjugate formation and immunofluorescence experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor cell conjugate formation (79, 80), Raji B cells (1 x 10\u003csup\u003e5\u003c/sup\u003e cells per coverslip) were washed once with HBSS and loaded with the CMAC cell tracker (10 \u0026mu;M; Molecular Probes) and with SEE (0.5 \u0026mu;g/mL; Toxin Technologies) for 1 h at 37\u0026deg;C in incomplete RPMI. Raji B cells were then washed twice with complete RPMI, conjugated with JK T cells (ratio 1:1) and then attached to poly-L-Lys-coated coverslips for 10 min (for K40-\u0026alpha;-tubulin acetylation determination and integrin activation assays), for 15 min (for mitochondrial membrane potential determination using MitoTracker Orange/Tom20 MFI ratio) or for 30 min (for centrosome and organelle polarization assays) at 37\u0026deg;C. For LFA-1 and VLA-4 integrins active conformation detection by IF, m24, KIM127 or HUTS-21 mAbs were added during JK-Raji cell conjugation prior to fixation, to avoid epitope loss during the fixation process. Negative controls were Raji B cells unloaded with SEE and conjugated with JK cells. CD4 T cells were incubated for 10 min (for K40-\u0026alpha;-tubulin acetylation determination) or 30 min (for centrosome and organelle polarization assays) at 37\u0026deg;C on coverslips coated with \u0026alpha;CD3 (15 \u0026micro;g/mL, clone HIT3a) and \u0026alpha;CD28 (5 \u0026micro;g/mL, clone CD28.2) or conjugated with anti-CD3 (20 \u0026mu;g/mL; clone HIT3a) and anti-CD28 (6.67 \u0026mu;g/mL; clone CD28.2) coated latex microbeads (6.4 \u0026mu;m in diameter, Sigma Aldrich) for 30 min at 37\u0026deg;C and were allowed to spread over poly-L-Lys plus fibronectin-coated coverslips. Negative controls were CD4 T cells conjugated with 100 \u0026mu;g/mL human \u0026gamma;-globulin-coated beads or settled over 50\u0026thinsp;\u0026micro;g/mL fibronectin-coated-coverslips. Cells were then fixed with 4% paraformaldehyde in PHEM (PIPES 30 mM, HEPES 20 mM, EGTA 2 mM, MgCl\u003csub\u003e2\u003c/sub\u003e 1 mM, pH: 6.9) containing 0.12 M sucrose for 10 min (R/T), permeabilized with TX-100 (0.2%) in PHEM for 5 min at R/T and blocked with PHEM containing 100 \u0026mu;g/mL \u0026gamma;-globulin, 3% BSA, 0.2% azide for 30 min at R/T. Cells were sequentially stained with the indicated primary antibodies (1-10 \u0026mu;g/mL) followed by Alexa Fluor 488-, 568- or 647-conjugated secondary antibodies (4 \u0026mu;g/mL), Alexa-conjugated phalloidin (5 \u0026mu;g/mL) or FITC-conjugated anti-\u0026alpha;-tubulin (0.1 \u0026mu;g/mL). Samples were mounted on Prolong gold or Prolong gold-DAPI (Invitrogen). A series of fluorescence and brightfield images were captured using a TCS SP5 confocal laser scanning unit (Leica Microsystems) attached to an inverted epifluorescence microscope (DMI6000) fitted with an HCX PL APO 63x/1.40-0.6 oil objective or a Leica STELLARIS Navigator confocal microscope equipped with a pulsed WLL (range, 470\u0026ndash;670\u0026thinsp;nm) and an HC PL Apo CS2 100\u0026times;/1.4 oil objective (Leica Microsystems). Epifluorescence images from CD4 T cells conjugated with latex microbeads were acquired as a Z-series of fluorescence and brightfield images under a THUNDER Imager Live Cell \u0026amp; 3D Cell Culture \u0026amp; 3D Assay and processed with the accompanying thunder algorithm for deconvolution (Leica Microsystems). A 100x objective was used.\u003c/p\u003e\n\u003cp\u003eImages were processed and analyzed using Image J software (http://rsbweb.nih.gov/ij/) and IMARIS software (Bit-plane) (https://imaris.oxinst.com). The \u003cem\u003e\u0026lsquo;Synapse Measures\u0026rsquo; \u003c/em\u003eplugin (http://rsbweb.nih.gov/ij/) was used to quantify mitochondria, peroxisomes or LD accumulation at the contact area\u003csup\u003e \u003c/sup\u003e(81). This program provides accurate measurements of localized immunofluorescence by comparing fluorescence signals from multiple regions of the T cell, APC, IS and after subtraction of background fluorescence. Maximal projections and 3D analysis of the T cell-APC contact area were generated using \u003cem\u003e\u0026lsquo;Z-project\u0026rsquo;, \u0026lsquo;Reslice\u0026rsquo;, \u0026lsquo;Plot profile\u0026rsquo; \u003c/em\u003eand\u003cem\u003e \u0026lsquo;3D surface plot\u0026rsquo; \u003c/em\u003efunctions of Image J. Colocalization was measured by using the built-in tool \u003cem\u003e\u0026lsquo;Colocalization threshold\u0026rsquo; \u003c/em\u003eand representing the Pearson\u0026rsquo;s or Manders coefficient. The distance of the centrosome or Golgi complex to the IS was calculated using IMARIS software (v8.4) by calculating volumes based on MFI and using the matlab implemented utility \u003cem\u003e\u0026lsquo;spots to volume distance\u0026rsquo;\u003c/em\u003e\u003csup\u003e \u003c/sup\u003e(82).\u003c/p\u003e\n\n\u003cp\u003ePreparation of stimulatory surfaces for TIRFm\u003c/p\u003e\n\u003cp\u003eGlass-bottom-18-well chambers (81817; Ibidi) were coated with 50 \u0026micro;L fibronectin (50\u0026thinsp;\u0026micro;g/mL) for 3 h at 37\u0026deg;C followed by 50 \u0026micro;L of anti-CD3\u0026epsilon; (20 \u0026mu;g/mL; HIT3a clone) and anti-CD28 (6.67 \u0026mu;g/mL; CD28.2 clone) monoclonal antibodies previously diluted in bicarbonate buffer (0.1 M NaHCO\u003csub\u003e3\u003c/sub\u003e and 0.32 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e) o/n at 4\u0026ordm;C. Before imaging, chambers were washed three times with HBSS, covered with 200 \u0026micro;L of imaging medium (HBSS supplemented with 2% FBS and 25 mM HEPES) and stored at 37\u0026deg;C until use.\u003c/p\u003e\n\n\u003cp\u003eLive-cell TIRFm acquisition and image analysis of microtubule dynamics \u003c/p\u003e\n\u003cp\u003eFor total internal reflection fluorescence microscopy (TIRFm), control and \u003cem\u003eSSH1\u003c/em\u003e KO cells were transfected in a Gene-Pulse III system (Bio-Rad) to overexpress EB3-GFP. Cells were incubated for 24 h, washed, and resuspended in imaging medium (1 x 10\u003csup\u003e6\u003c/sup\u003e cells/100 \u0026mu;l). Then, 20 \u0026micro;L (2 x 10\u003csup\u003e5\u003c/sup\u003e cells) were seeded onto glass-bottom-18-well chamber coated with stimulatory anti-CD3\u0026epsilon; and anti-CD28 monoclonal antibodies. Imaging was performed using Leica AM TIRF MC M system mounted on a Leica DMI 6000B fitted with a HCX PL APO 100x1.46 NA oil objective microscope, coupled to an Andor-DU8285 VP-4094 camera. EB3-GFP was excited with the 488 nm laser at 2-5% laser power. Frames were acquired every 300 ms for 3 min and 100-200 ms of exposure time, with a Z penetrance of 150 nm. Synchronization was performed with the accompanying Leica software, and images were analyzed using \u0026ldquo;\u003cem\u003eTrackMate\u003c/em\u003e\u0026rdquo; plugin from ImageJ software (http://rsbweb.nih.gov/ij/). Laplacian of Gaussian (LoG Detector) and Linear Assignment Problem (LAP) tracker were used. To detect the EB3-GFP tips, the following values were used: \u003cem\u003eEstimated object diameter\u003c/em\u003e: 0.5 \u0026micro;m. \u003cem\u003eLinking Max distance\u003c/em\u003e: 0.5 \u0026micro;m; \u003cem\u003eGap-closing Max frame gap\u003c/em\u003e: 1\u0026ndash;2 frames; \u003cem\u003eGap-closing Max distance\u003c/em\u003e: 1.0 \u0026micro;m. Speed, number of tracks and displacement were calculated.\u003c/p\u003e\n\u003cp\u003eTrackMate outputs were post-processed using a Python pipeline to compute per-cell microtubule growth descriptors. Code and a detailed documentation are available in the repository instructions (https://github.com/MLozanoPrieto/trackmate-eb3-tirfm-analysis).\u003c/p\u003e\n\n\u003cp\u003eFlow cytometry staining\u003c/p\u003e\n\u003cp\u003e1-3 x 10\u003csup\u003e5\u003c/sup\u003e cells of each cellular type were employed in each flow cytometry staining. Primary and secondary antibody staining was maintained for 30 min on ice and washed with FACS buffer (HBSS, 50 \u0026micro;g/mL human \u0026gamma;-globulin, 2% BSA, 1 mM EDTA). For LFA-1 and VLA-4 integrins active conformation detection by flow cytometry, 3-5 x 10\u003csup\u003e5\u003c/sup\u003e human primary CD4 T cells or JK T cells were activated in 96-well plates coated with \u0026alpha;CD3 (20 \u0026micro;g/mL, clone HIT3a) and \u0026alpha;CD28 (6.67 \u0026micro;g/mL, clone CD28.2) for 15 min and simultaneously incubated with m24, KIM127 or HUTS-21 mAbs, to detect the active conformation of LFA-1 and VLA-4, respectively. Then, cells were washed three times with HBSS and stained with goat anti-mouse IgG (H+L) secondary antibody conjugated to PE (1:500; ThermoFisher) for 30 min on ice. Finally, cells were washed twice with FACS buffer and resuspended in 200 \u0026mu;L of FACS buffer for flow cytometry acquisition. For peroxisome detection by flow cytometry, control and \u003cem\u003eSSH1\u003c/em\u003e KO JK T cells were fixed with fixation buffer (420801; BioLegend) for 30 min on ice, washed with permeabilization buffer (421002; BioLegend) and incubated with anti-Pex14 rabbit antibody (1:100; ThermoFisher) for 30 min on ice followed by goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (1:500; ThermoFisher) in permeabilization buffer. Finally, cells were washed with permeabilization buffer and resuspended in 200 \u0026mu;L of FACS buffer for flow cytometry acquisition. Data were acquired using a FACS Canto II analyzer cytometer (405 nm violet laser, 488 nm solid state blue laser and 633 nm He-Ne) (BD Biosciences) and analyzed using FlowJo software (v10.7) (BD Biosciences).\u003c/p\u003e\n\n\u003cp\u003eIsolation of mitochondria for Western blot analysis\u003c/p\u003e\n\u003cp\u003e1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e control or \u003cem\u003eSSH1\u003c/em\u003e KO Jurkat T cells were stimulated or not for 20 min with 50 \u0026mu;L \u0026alpha;CD3/\u0026alpha;CD28 antibodies (ImmunoCult Human CD3/CD28 T cell Activator) in 1 mL of incomplete RPMI and mitochondria were isolated using a human mitochondria isolation kit following the manufacturer\u0026rsquo;s instructions (130-094-833; Miltenyi Biotec). Isolated mitochondria were resuspended in 50 \u0026mu;L of RIPA buffer with phosphatase and protease inhibitors (Phosphostop and Complete tablets from Roche, respectively), then sonicated for 1 h and processed for SDS-PAGE in 8 % polyacrylamide gels, transferred to nitrocellulose membranes and subjected to Western blotting. Total lysates were extracted prior to mitochondrial lysis and isolation.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMitochondrial mass, mitochondrial ROS and mitochondrial membrane potential quantification through flow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial mass was assessed in control and \u003cem\u003eSSH1\u003c/em\u003e KO Jurkat or in control or SSH1-silenced human primary CD4 T cells by labeling with nonyl-acridine orange (NAO, 25 nM; Life Technologies-Invitrogen) for 20 min at 37\u0026ordm;C and 5% CO\u003csub\u003e2\u003c/sub\u003e followed by CD4-APC in FACS buffer for 20 min at 4\u0026deg;C. Mitochondrial ROS was determined in control or SSH1-silenced human primary CD4 T cells by labeling with MitoSOX Green (M36006; 1 \u0026micro;M) for 30 min at 37\u0026ordm;C and 5% CO\u003csub\u003e2\u003c/sub\u003e and then stimulated over 96-well plates coated with anti-CD3 (20 \u0026mu;g/mL; clone HIT3a) and anti-CD28 (6.67 \u0026mu;g/mL; clone CD28.2) antibodies for the indicated times. Cells were then stained with Ghost dye Violet 780 Viability Dye (1:1,000, Tonbo Biosciences) in 100 \u0026micro;L of PBS for 30 min at 4\u0026deg;C and CD4-PE for 20 min at 4\u0026deg;C in FACS buffer. Mitochondrial membrane potential was assessed in control or SSH1-silenced human primary CD4 T cells by labeling with MitoTracker Deep Red FM (M22426, 500 nM) for 30 min at 37\u0026ordm;C and 5% CO\u003csub\u003e2\u003c/sub\u003e and then stimulated over 96-well plates coated with anti-CD3 (20 \u0026mu;g/mL; clone HIT3a) and anti-CD28 (6.67 \u0026mu;g/mL; clone CD28.2) antibodies for the indicated times. Cells were then stained with Ghost dye Violet 780 Viability Dye (1:1,000, Tonbo Biosciences) in 100 \u0026micro;L of PBS for 30 min at 4\u0026deg;C and CD4-PE for 20 min at 4\u0026deg;C in FACS buffer. Finally, cells were washed with FACS buffer and resuspended in 100 \u0026micro;L of FACS buffer for flow cytometry acquisition. Mean fluorescence intensity and geometric mean of staining were acquired using a FACS Canto II analyzer cytometer (405 nm violet laser, 488 nm solid state blue laser and 633 nm He-Ne) (BD Biosciences) and analyzed using FlowJo software (v10.7) (BD Biosciences).\u003c/p\u003e\n\n\u003cp\u003eReal-time cell metabolic analysis using Seahorse \u003c/p\u003e\n\u003cp\u003eThe oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured using an XF Pro extracellular flux analyzer (Seahorse Bioscience; XFPro M FluxPak Agilent Technologies). For the glucose-based mitostress test, the use of glucose was measured in control and \u003cem\u003eSSH1\u003c/em\u003e KO Jurkat or SSH1-silenced human primary CD4 T cells, in basal conditions or after the stimulation by injection of ImmunoCult Human CD3/CD28 T Cell Activator (Stem Cell Technologies). Cells were cultured with Dulbecco\u0026rsquo;s modified Eagle medium (DMEM) (D5030, Sigma Aldrich) supplemented with 1 mM sodium pyruvate, 1 mM L-glutamine, and 25 mM glucose and were seeded at 0.3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e Jurkat T cells or 0.5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e in human primary CD4 T cells per well in culture plates pre-coated with poly-L-Lys (50\u0026thinsp;\u0026micro;g/mL). Each plate included four independent donors in the case of SSH1-silenced human primary CD4 T cells and five technical replicates. Drugs were injected as follows: oligomycin (1.8 \u0026mu;M), CCCP (2 \u0026mu;M), rotenone (1 \u0026mu;M), and antimycin A (1 \u0026mu;M). Three consecutive mix and measure steps were performed for resting conditions and after each injection (3 min each). For the glycolysis stress assay, cells were cultured with DMEM supplemented with 2 mM L-glutamine and seeded as before (four biological and five technical replicates per plate). Injections were as follows: glucose (10 mM), oligomycin (1.8 \u0026mu;M), and 2-deoxyglucose (2-DG; 50 mM). Mix and measure steps were as before. For the palmitate-based mitostress test, to evaluate the FAO rate, control and \u003cem\u003eSSH1\u003c/em\u003e KO Jurkat or SSH1-silenced human primary CD4 T cells were cultured in substrate-limited growth media (DMEM medium containing 1% FCS, 2.5 mM glucose, 25 mM HEPES, 0.5 mM L-carnitine and 1 mM L-glutamine). The day of the experiment, cells were washed and seeded at 0.3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e Jurkat T cells or 0.5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e in human primary CD4 T cells per well in FAO assay media (111 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 1.2 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 1 mM glucose, 0.5 mM L-carnitine, 5 mM HEPES, 125 \u0026mu;M Palmitate-BSA or BSA as control, 40 \u0026mu;M etomoxir or media as control) on poly-L-Lys pre-coated culture plate from XF96 FluxPak (Agilent Technologies). Drugs were injected as follows: medium or ImmunoCult Human CD3/CD28 T Cell Activator, oligomycin (2 \u0026mu;M), CCCP (2 \u0026mu;M), rotenone (1 \u0026mu;M) plus antimycin A (1 \u0026mu;M). Mix and measure steps were as before. Seahorse results were analyzed using Seahorse Wave Pro software. Statistical analysis of time-course OCR/ECAR data was performed in R (v4.2.1) (https://cran.r-project.org) (see Supplementary information). For control and \u003cem\u003eSSH1\u003c/em\u003e KO Jurkat T cells, values were analyzed using linear models (value ~ treatment \u0026times; condition) followed by estimated marginal means and within-block pairwise contrasts (emmeans; Tukey adjustment). For siCTRL and siSSH1 primary CD4 T cells, data were averaged within each injection block and across technical replicates to obtain one mean per donor \u0026times; condition \u0026times; block and analyzed on the log scale with donor as a blocking factor, using HC3 heteroscedasticity-robust standard errors and Holm-adjusted within-block contrasts (emmeans; back-transformed).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipid droplet and lipid peroxidation quantification through flow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor LD quantification and lipid peroxidation determination, control and \u003cem\u003eSSH1\u003c/em\u003e KO Jurkat T cells were stained with BODIPY 493/503 (0.8 \u0026mu;M; ThermoFisher) or BODIPY 581/591 C11 (0.8 \u0026mu;M; ThermoFisher) in HBSS (Lonza) for 30 min at 37\u0026deg;C in incomplete RPMI, respectively, and LIVE/DEAD Fixable Blue dead cell stain (1:1,000; L23105; ThermoFisher) according to manufacturer\u0026apos;s instructions and analyzed by flow cytometry. Lipid peroxidation was obtained as the MFI in the B530/30 channel (peroxidation signal with an emission wavelength of \u0026sim;510 nm) of viable cells or the fluorometric ratio between the B530/30 and YG586/15 channels (basal signal with emission wavelength of \u0026sim; 590 nm) as described (83), using a FACS Symphony SORP analyzer (BD Biosciences). Data were analyzed using FlowJo software (v10.7) (BD Biosciences).\u003c/p\u003e\n\n\u003cp\u003eStatistics and reproducibility\u003c/p\u003e\n\n\u003cp\u003eStatistical analyses were performed using PRISM8 (GraphPad software). Normality was assessed with Shapiro-Wilk test. Statistical analyses were performed using parametric Student\u0026rsquo;s t-test, two-tailed paired t-test, and one-way or two-way ANOVA with Bonferroni multiple comparison test, as indicated. Mann-Whitney U and Kruskal-Wallis were used for non-parametric analyses. When comparing two or more samples, including time courses, two-way ANOVA was used. Sample sizes and specific details of each analysis are indicated in the figure legends. Significant differences were considered at p\u0026lt;0.05. Statistical differences are indicated as asterix (*p \u0026le; 0.05, **p \u0026le; 0.01, ***p \u0026le; 0.001, ****p \u0026le; 0.0001) or non-significant (ns). The schematics and figures were generated with Adobe Illustrator (Adobe, v2020).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAGM\u003c/strong\u003e, conceptualization, experimental design and execution, data curation (cell biology, transfection/nucleofection, flow cytometry, immunoprecipitation assays, Western blot, TIRFm and confocal microscopy), image composition, writing (original draft, review and editing), Fig. 1-5; Supplementary Fig. 1-6; \u003cstrong\u003eMLP\u003c/strong\u003e, data curation (TIRFm and Seahorse statistical analysis), writing (review and editing), Fig. 3,4; Supplementary Movie1; \u003cstrong\u003eCS\u003c/strong\u003e, \u003cstrong\u003eCCP\u003c/strong\u003e, \u003cstrong\u003eOAS \u003c/strong\u003eand \u003cstrong\u003eLMA \u003c/strong\u003eperformed experiments (flow cytometry, microscopy), Fig. 1-2; \u003cstrong\u003eFSM \u003c/strong\u003eresources, funding acquisition, data curation, revised manuscript; \u003cstrong\u003ePRN \u003c/strong\u003eand \u003cstrong\u003eNBMC \u003c/strong\u003eplanned and coordinated research, conceptualization, resources, funding acquisition, data curation and interpretation, writing (review and editing). All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from Madrid Regional Government (S2022/BMD-7209-INTEGRAMUNE-CM) to NBMC, from Spanish Ministry of Science and Innovation funded by MCIN/AEI/10.13039/501100011033 in part granted with ERDF \u0026ldquo;A way of making Europe\u0026rdquo; (PID2022-141895OB-I00) to NBMC, (PID2023-147805NB-I00) to PRN, (PID2023-149541OB-I00) to FSM. NBMC and FSM are also funded by Fundaci\u0026oacute;n LaCaixa (LCF/PR/HR23/52430018) and CIBER Cardiovascular (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III and co-funded by Fondo Europeo de Desarrollo Regional FEDER). AGM (PIPF-2023/SAL-GL-30092) and CCP (PIPF-2022/SAL-GL-24353) are supported by a PhD Fellowship from the Madrid Regional Government. MLP is supported by an FPI fellowship (PRE2021-097478). CS (PEJ-2021-TL/BMD-21204) and LMA (PEJ-2024-TL/SAL-GL-33552) are supported by \u0026ldquo;Garantı́a Juvenil\u0026apos;\u0026apos; grant to NBMC from Comunidad de Madrid. OAS is funded by a PhD fellowship of Universidad Complutense de Madrid. Funding agencies have not intervened in the design of the studies, with no copyright over the study. Optical microscopy experimentation was conducted at (1) the Microscopy \u0026amp; Dynamic Imaging, CNIC, ICTS-ReDib, cofunded by MCIN/AEI/10.13039/501100011033 and FEDER Una manera de hacer Europa\u0026rdquo; (#ICTS-2018-04-CNIC-16) and (2) the Videomicroscopy Facility of the IIS-IP (Madrid, Spain), co-funded by IFEQ21/00085 and IFCS22/00014 from ISCIII and FEDER. We are grateful to Ms. M \u0026Aacute;ngeles Vallejo and Ana Cayuela for her helpful assistance and management.\u003c/p\u003e\n\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe data underlying this article are available in the article and in its Supplementary Information. Numerical data source is provided in the Supplementary data. All other data and source data are available from the corresponding authors on reasonable request.\u003c/p\u003e\n\n\u003cp\u003eInclusion and ethics approval\u003c/p\u003e\n\u003cp\u003eThese studies were performed according to the principles of the Declaration of Helsinki and approved by the local Ethics Committee for Basic Research at the \u003cem\u003eHospital La Princesa \u003c/em\u003e(Madrid).\u003c/p\u003e\n\u003cp\u003eConsent to publish\u003c/p\u003e\n\u003cp\u003eConsent to publish has been received from all participants.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eG\u0026oacute;mez-Mor\u0026oacute;n, \u0026Aacute;., Gonz\u0026aacute;lez-Pinar, A., Carrasco-Padilla, C., Roda-Navarro, P., \u0026amp; Mart\u0026iacute;n-C\u0026oacute;freces, N. B. (2025). Exploring How Cytoskeleton Dynamics Tunes T Cell Activation at the Immunological Synapse. 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Nature communications, 14(1), 6772. https://doi.org/10.1038/s41467-023-42480-3\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Slingshot-1, T lymphocytes, metabolism, mitochondria, cytoskeleton, integrin activation, Immunological Synapse","lastPublishedDoi":"10.21203/rs.3.rs-8604514/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8604514/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Effective T cell activation requires formation of the immunological synapse (IS), which depends on coordinated remodeling of actin and microtubule cytoskeletons. Upon T cell receptor (TCR) engagement, the phosphatase Slingshot-1 (SSH1) is rapidly recruited to the nascent IS, where it dephosphorylates cofilin and suppresses LIM kinase activity to promote actin dynamics. However, the mechanism by which SSH1 anchors at and controls IS formation is unknown. Here, we identify the role of SSH1 in assembling a protein hub with Talin-1, Kindlin-3, ADAP, and Myosin IIA that promotes high-affinity activation of the integrin LFA-1. This allows SSH1 to coordinately regulate actin and microtubule dynamics and ultimately facilitates the metabolic reprogramming of T cells. Loss of SSH1 disorganizes IS in terms of mitochondria, rewiring T cells toward fatty acid oxidation, congregating lipid droplets and peroxisomes, and preventing glucose metabolism. Together, our findings establish SSH1 as a node connecting cytoskeletal dynamics to metabolic adaptation for T cell activation.","manuscriptTitle":"Slingshot-1 (SSH1) phosphatase Controls Cytoskeletal Remodeling, Integrin conformation and Metabolic Reprogramming During CD4 T Cell Activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-14 10:32:35","doi":"10.21203/rs.3.rs-8604514/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"afb98d24-715b-440b-bea4-f8653edb7aee","owner":[],"postedDate":"April 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61984241,"name":"Biological sciences/Cell biology/Cell adhesion/Integrins"},{"id":61984242,"name":"Biological sciences/Immunology/Imaging the immune system"},{"id":61984243,"name":"Biological sciences/Cell biology/Cytoskeleton/Actin"},{"id":61984244,"name":"Biological sciences/Immunology/Adaptive immunity/Cellular immunity/Lymphocyte activation"},{"id":61984245,"name":"Biological sciences/Biochemistry/Lipids/Fatty acids"}],"tags":[],"updatedAt":"2026-04-14T10:32:36+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-14 10:32:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8604514","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8604514","identity":"rs-8604514","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}